Hypersensitive response elicitor peptides and use thereof

ABSTRACT

Disclosed are hypersensitive-response eliciting peptides that exhibit improved solubility, stability, resistance to chemical degradation, or a combination of these properties. Use of these peptides or fusion polypeptides, or DNA constructs encoding the same, for modulating plant biochemical signaling, imparting disease resistance to plants, enhancing plant growth, imparting tolerance to biotic stress, imparting tolerance and resistance to abiotic stress, imparting desiccation resistance to cuttings removed from ornamental plants, imparting post-harvest disease or post-harvest desiccation resistance to a fruit or vegetable, or enhancing the longevity of fruit or vegetable ripeness are also disclosed.

This application claims the priority benefit of U.S. Provisional PatentApplication Ser. No. 62/058,535, filed Oct. 1, 2014, and U.S.Provisional Patent Application Ser. No. 62/140,789, filed Mar. 31, 2015,each of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates to novel hypersensitive response elicitorpeptides and their use for inducing active plant responses including,among others, growth enhancement, disease resistance, pest or insectresistance, and stress resistance.

BACKGROUND OF THE INVENTION

The identification and isolation of harpin proteins came from basicresearch at Cornell University attempting to understand how plantpathogenic bacteria interact with plants. A first line of defense is thehypersensitive response (HR), a localized plant cell death at the siteof infection. Cell death creates a physical barrier to movement of thepathogen and in some plants dead cells can release compounds toxic tothe invading pathogen. Research had indicated that pathogenic bacteriawere likely to have a single factor that was responsible for triggeringthe HR. A basic aim of the Cornell research was to identify a specificbacterial protein responsible for eliciting the HR. The target proteinwas known to be encoded by one of a group of bacteria genes called theHypersensitive Response and Pathogenicity (hrp) gene cluster. The hrpcluster in the bacterium Erwinia amylovora (Ea), which causes fireblight in pear and apple, was dissected and a single protein wasidentified that elicited HR in certain plants. This protein was giventhe name harpin (and, later, harpin_(Ea)) and the corresponding genedesignated hrpN. This was the first example of such a protein and geneidentified from any bacterial species.

A number of different harpin proteins have since been identified fromErwinia, Pseudomonas, Ralstonia, Xanthomonas, and Pantoea species, amongothers. Harpin proteins, while diverse at the primary amino acidsequence level, share common biochemical and biophysical characteristicsas well as biological functions. Based on their unique properties, theharpin proteins are regarded in the literature as belonging to a singleclass of proteins.

Subsequent to their identification and isolation, it was thereafterdiscovered that harpins could elicit disease resistance in plants andincrease plant growth. An important early finding was that applicationof purified harpin protein made a plant resistant to a subsequentpathogen attack, and in locations on the plant well away from theinjection site. This meant that harpin proteins can trigger a SystemicAcquired Resistance (SAR), a plant defense mechanism that providesresistance to a variety of viral, bacterial, and fungal pathogens.

In crop protection, there is a continuous need for compositions thatimprove the health of plants. Healthier plants are desirable since theyresult in better yields and/or a better quality of the plants or crops.Healthier plants also better resist biotic and abiotic stress. A highresistance against biotic stresses in turn allows the growers to reducethe quantity of pesticides applied and consequently to slow down thedevelopment of resistances against the respective pesticides.

Harpin_(αβ) is a fusion protein that is derived from several differentharpins. Harpin_(αβ) has been shown to suppress nematode egg production,enhance the growth, quality and yield of a plant, and increase a plant'svigor. Its amino acid and nucleotide sequences are described in detailin U.S. Application Publ. No. 2010/0043095.

To date, harpin and harpin_(αβ) production and their use in agriculturaland horticultural applications have been as a powdered solid coated onstarch. This limits the use and versatility of the harpin proteins,because liquid suspensions of the powdered harpin proteins in water havean effective useful life of only 48-72 hours before significantdegradation and loss of activity occurs. Another problem with harpinsolutions is protein solubility and stability.

It would be desirable to identify synthetic and derivative harpinpeptides that are readily soluble in aqueous solution, stable, resistantto chemical degradation, and effective in initiating the hypersensitiveresponse in plants.

The present invention is directed to overcoming these and otherlimitations in the art.

SUMMARY OF THE INVENTION

A first aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:(L/I/V/F)-X—X-(L/I/V/F)-(L/I)-X—X-(L/I/V/F)-(L/I/V/A)-X—X-(L/I)-(L/I/V/F)(SEQ ID NO: 93) wherein the peptide is free of cysteine and methionine;each X at positions 2 and 6 is optional and, when present, is any aminoacid; and each X at positions 3, 7, 10, and 11 is any amino acid. In oneembodiment, X at only one of positions 2 and 6 is optional. In certainembodiments, SEQ ID NO: 93 may further include an additional amino acidresidue between the hydrophobic doublets (two of L/I/V/F/A, asindicated). In certain embodiments, the isolated peptide furtherincludes a hydrophilic amino acid sequence that is located N-terminal orC-terminal to SEQ ID NO: 93.

A second aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:(L/I/V/F)-X—X-(L/I/V/F)-(L/I)-X—X-(L/I/V/F)-(L/I/V/A)-X—X-(L/I)-(L/I/V/F)(SEQ ID NO: 93) wherein the peptide is free of cysteine and methionine;each X at positions 2, 6, and 10 is optional and, when present, is anyamino acid; and each X at positions 3, 7, and 11 is any amino acid. Inone embodiment, X at only one of positions 2, 6, and 10 is optional. Incertain embodiments, SEQ ID NO: 93 may further include an additionalamino acid residue between the hydrophobic doublets (two of L/I/V/F/A,as indicated). In certain embodiments, the isolated peptide furtherincludes a hydrophilic amino acid sequence that is located N-terminal orC-terminal to SEQ ID NO: 93.

A third aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

XXGISEKXXXXXXXXXXXXXXXX (SEQ ID NO: 1, P1/P4 consensus), wherein

-   -   X at position 1 is optional and can be S, N, D, isoD, G, A, or        S;    -   X at position 2 is optional and can be Q, E, g-glutamate, G, A,        or S;    -   X at position 8 is Q, E, g-glutamate, G, A, or S;    -   X at position 9 is L, I, F, or V;    -   X at position 10 is optional and can be D or isoD;    -   X at position 11 is Q, E, g-glutamate, G, A, or S;    -   X at position 12 is M, L, I, or F;    -   X at position 13 is M, L, or I;    -   X at position 14 is optional and can be any hydrophilic amino        acid, preferably C, S, T, A, D, isoD, K, or Q;    -   X at position 15 is Q, E, g-glutamate, G, A, S, K, or I;    -   X at position 16 is M, L, I, V, or F;    -   X at position 17 is M, L, I, A, or V;    -   X at position 18 is Q, E, g-glutamate, G, A, S, M, T, or K;    -   X at position 19 is A, D, isoD, S, V, T, K, R, E, H, or G;    -   X at position 20 is M, L, or I;    -   X at position 21 is M, L, I, V, S, or F;    -   X at position 22 is Q, E, g-glutamate, G, A, S;    -   X at position 23 is P, Q, E, g-glutamate, G, A, or S; and        wherein the isolated peptide comprises one or more mutations        relative to a corresponding wildtype amino acid sequence. In        certain embodiments, the one or more mutations improve the        aqueous solubility, stability, or resistance to chemical        degradation of the isolated peptide relative to a polypeptide        comprising the corresponding wildtype amino acid sequence.

One exemplary family of peptides according to the third aspect of theinvention have the amino acid sequence of:

SXGISEKXXDXXXXXXXXAXXXP (SEQ ID NO: 2, P4 consensus), wherein

-   -   X at position 2 is Q, E, g-glutamate, G, A, or S;    -   X at position 8 is Q, E, g-glutamate, G, A, or S;    -   X at position 9 is L, A, D, isoD, I, V, or F;    -   X at position 11 is Q, E, g-glutamate, G, A, or S;    -   X at position 12 is L, D, isoD, I, or F;    -   X at position 13 is L, I, V, or F;    -   X at position 14 is any hydrophilic amino acid, preferably C, S,        or T, S or T, or only S;    -   X at position 15 is Q, E, g-glutamate, G, A, S, K, or I;    -   X at position 16 is L, A, I, V, M, or F;    -   X at position 17 is I, S, or F;    -   X at position 18 is Q, E, g-glutamate, G, A, or S;    -   X at position 20 is L, I, V, or F;    -   X at position 21 is L or F; and    -   X at position 22 is Q, E, g-glutamate, G, A, or S.        In certain embodiments, these peptides according to the third        aspect of the invention also meet the structural features        defining the peptides according to the first or second aspect of        the invention.

Another exemplary family of peptides according to the third aspect ofthe invention have the amino acid sequence of:

XXGISEKXLDXLLTXLIXALLXX (SEQ ID NO: 3, P1 consensus), wherein

-   -   X at position 1 is N, D, isoD, G, A, or S;    -   X at position 2 is Q, E, g-glutamate, G, A, or S;    -   X at position 8 is Q, E, g-glutamate, G, A, or S;    -   X at position 11 is Q, E, g-glutamate, G, A, or S;    -   X at position 15 is Q, E, g-glutamate, G, A, or S;    -   X at position 18 is M, T, K, E, g-glutamate, G, A, or S;    -   X at position 22 is Q, E, g-glutamate, G, A, or S; and    -   X at position 23 is Q, E, g-glutamate, G, A, or S.        In certain embodiments, these peptides according to the third        aspect of the invention also meet the structural features        defining the peptides according to the first or second aspect of        the invention.

A fourth aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

-   (i) KPXDSXSXIAKLISXLIXSLLX (SEQ ID NO: 47, P15b/P20 consensus),    wherein    -   X at position 3 is N, D, or isoD;    -   X at position 6 is Q, E, g-glutamate, G, A, or S;    -   X at position 8 is N, D, or isoD;    -   X at position 15 is optional and can be any amino acid;    -   X at position 18 is M, E, g-glutamate, G, A, S, T, or K; and    -   X at position 22 is optional and can be Q, E, g-glutamate, G, A,        or S; or-   (ii) IAKLISXLIXSLLX (SEQ ID NO: 12, P15/20 min consensus), wherein    -   X at position 7 is optional and can be any amino acid;    -   X at position 10 is M, E, g-glutamate, G, A, S, T, or K; and    -   X at position 14 is optional and can be Q, E, g-glutamate, G, A,        or S.        In certain embodiments, the isolated peptide comprises one or        more mutations relative to a corresponding wildtype amino acid        sequence, and the one or more mutations improve the aqueous        solubility, stability, or resistance to chemical degradation of        the isolated peptide relative to a polypeptide comprising the        corresponding wildtype amino acid sequence. In certain        embodiments, the peptides according to the fourth aspect of the        invention also meet the structural features defining the        peptides according to the first or second aspect of the        invention.

A fifth aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

-   (i) PSPXTXXLXXIVGXILXAXN (SEQ ID NO: 66, P6/6a consensus), wherein    -   X at position 4 is F or Y;    -   X at position 6 is Q, E, g-glutamate, G, A, or S;    -   X at position 7 is optional and can be L, M, E, g-glutamate, G,        A, S, T, or K;    -   X at position 9 is M, E, g-glutamate, G, A, S, T, or K;    -   X at position 10 is H or N;    -   X at position 14 is E, g-glutamate, D, or isoD;    -   X at position 17 is Q, E, g-glutamate, G, A, or S; and    -   X at position 19 is Q, E, g-glutamate, G, A, or S; or-   (ii) XTXXLXXIVGXIL (SEQ ID NO: 135, P6/6a min consensus), wherein    -   X at position 1 is F or Y;    -   X at position 3 is Q, E, g-glutamate, G, A, or S;    -   X at position 4 is optional and, according to one embodiment,        can be M, E, g-glutamate, G, A, S, T, or K; or according to        another embodiment can be L;    -   X at position 6 is M, E, g-glutamate, G, A, S, T, or K;    -   X at position 7 is H or N; and    -   X at position 11 is E, g-glutamate, D, or isoD;        wherein the isolated peptide comprises one or more mutations        relative to a corresponding wildtype amino acid sequence, and        the one or more mutations improve the aqueous solubility,        stability, or resistance to chemical degradation of the isolated        peptide relative to a polypeptide comprising the corresponding        wildtype amino acid sequence. In certain embodiments, the        peptides according to the fifth aspect of the invention also        meet the structural features defining the peptides according to        the first or second aspect of the invention.

A sixth aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

-   (i) LXXLLXXLVXLLK (SEQ ID NO: 13, P14d consensus), wherein    -   X at position 1 can be: Q, N, D, E, g-glutamate, isoD, or S;    -   X at position 2 can be: D, E, g-glutamate, isoD;    -   X at position 3 can be: P, D, E, isoD, or g-glutamate;    -   X at position 4 can be M, A, S, D, E, isoD, or g-glutamate    -   X at position 5 can be Q, E, or g-glutamate;    -   X at position 6 can be A, E, or g-glutamate;    -   X at position 8 can be M, L, E, Q, D, N, G, A, S, isoD, or        g-glutamate;    -   X at position 9 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 12 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 13 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate; and    -   X at position 16 can be K, Q, N, E, D, R, G, A, or S; or-   (ii) LXXLLXXLVXLLK (SEQ ID NO: 14, P14d min consensus), wherein    -   X at position 2 can be M, L, E, Q, D, N, G, A, S, isoD, or        g-glutamate;    -   X at position 3 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 6 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 7 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate; and    -   X at position 10 can be K, Q, N, E, D, R, G, A, or S.        In certain embodiments, the isolated peptide comprises one or        more mutations relative to a corresponding wildtype amino acid        sequence, and the one or more mutations improve the aqueous        solubility, stability, or resistance to chemical degradation of        the isolated peptide relative to a polypeptide comprising the        corresponding wildtype amino acid sequence. In certain        embodiments, the peptides according to the sixth aspect of the        invention also meet the structural features defining the        peptides according to the first or second aspect of the        invention.

A seventh aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

-   (i) LXXL(L/M)XILXXLV (SEQ ID NO: 16, P25 consensus), wherein    -   X at position 2 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G;    -   X at position 3 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 6 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 9 can be E, g-glutamate, D, isoD, Q, N, T, S, A,        or G; and    -   X at position 10 can be A, G, S, T, E, g-glutamate, D, isoD, Q,        or N; or-   (ii) LXXVLXXL(L/M)XILXXLV (SEQ ID NO: 17, P25 consensus), wherein    -   X at position 2 can be T, S, A, G, D, isoD, E, g-glutamate, Q,        or N;    -   X at position 3 can be G, T, S, A, D, isoD, E, g-glutamate, Q,        or N;    -   X at position 6 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G;    -   X at position 7 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 10 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 13 can be E, g-glutamate, D, isoD, Q, N, T, S, A,        or G;    -   X at position 14 can be A, G, S, T, E, g-glutamate, D, isoD, Q,        or N; and    -   V at position 16 is optional.        In certain embodiments, the isolated peptide comprises one or        more mutations relative to a corresponding wildtype amino acid        sequence, and the one or more mutations improve the aqueous        solubility, stability, or resistance to chemical degradation of        the isolated peptide relative to a polypeptide comprising the        corresponding wildtype amino acid sequence. In certain        embodiments, the peptides according to the seventh aspect of the        invention also meet the structural features defining the        peptides according to the first or second aspect of the        invention.

An eighth aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

-   (i) XXXXXXXXXXX(L/M)XXLLXXLLXXLLXXX (SEQ ID NO: 21, P17/18), wherein    -   X at position 1 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 2 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 3 can be any amino acid, but preferably P, Q, S,        E, g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 4 can be any amino acid, but preferably I, Q, S,        E, g-glutamate, A, T, G, D, N, isoD, K, or R;    -   X at position 5 can be any amino acid, but preferably D, isoD,        S, E, g-glutamate, A, T, G, N, Q, K, or R;    -   X at position 6 can be any amino acid, but preferably R, Q, S,        E, g-glutamate, A, T, G, D, isoD, N, or K;    -   X of position 7 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 8 can be any amino acid, but preferably T, Q, S,        E, g-glutamate, A, G, D, isoD, N, K, or R;    -   X at position 9 can be any amino acid, but preferably I, Q, S,        E, g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 10 can be any amino acid, but preferably E,        g-glutamate, Q, S, A, T, G, D, isoD, N, K, or R;    -   X at position 11 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 13 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 14 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 17 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 18 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 21 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 22 can be any amino acid, but preferably S, A,T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 25 can be any amino acid, but preferably S, A, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 26 can be any amino acid, but preferably P, S, A,        T, G, D, isoD, E, g-glutamate, Q, N, K, or R; and    -   X at position 27 can be any amino acid, but preferably Q, S, A,        T, G, D, isoD, E, g-glutamate, N, K, or R; or-   (ii) (L/M)XXLLXXLLXXLL (SEQ ID NO: 25, P17/18 min consensus),    wherein    -   X at position 2 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 3 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 6 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 7 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 10 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R; and    -   X at position 11 can be any amino acid, but preferably S, A, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;        wherein the isolated peptide comprises one or more mutations        relative to a corresponding wildtype amino acid sequence, and        the one or more mutations improve the aqueous solubility,        stability, or resistance to chemical degradation of the isolated        peptide relative to a polypeptide comprising the corresponding        wildtype amino acid sequence. In certain embodiments, the        peptides according to the eighth aspect of the invention also        meet the structural features defining the peptides according to        the first or second aspect of the invention.

A ninth aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

-   XLXX(L/M)LXLIXX(L/I/V/F/M)(L/I/V/F/M) (SEQ ID NO: 26, P19    consensus), wherein    -   X at position 1 is optional and can be L, I, V, F, or M;    -   X at position 3 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 4 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 7 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 10 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate,

Q, N, K, or R; and

-   -   X at position 11 can be any amino acid, but preferably R, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or K.        In certain embodiments, the isolated peptide comprises one or        more mutations relative to a corresponding wildtype amino acid        sequence, and the one or more mutations improve the aqueous        solubility, stability, or resistance to chemical degradation of        the isolated peptide relative to a polypeptide comprising the        corresponding wildtype amino acid sequence. In certain        embodiments, the peptides according to the ninth aspect of the        invention also meet the structural features defining the        peptides according to the first or second aspect of the        invention.

A tenth aspect of the invention relates to an isolated peptide thatincludes the amino acid sequence of

(L/M)XXLLX(L/M)FXXI(L/M)XX (SEQ ID NO: 15, P3min consensus) wherein

-   -   X at position 2 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G;    -   X at position 3 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G;    -   X at position 6 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 9 can be E, g-glutamate, D, isoD, Q, N, T, S, A,        or G;    -   X at position 10 can be A, G, S, T, E, g-glutamate, D, isoD, Q,        or N;    -   X at position 13 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G; and    -   X at position 14 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G.        In certain embodiments, the isolated peptide comprises one or        more mutations relative to a corresponding wildtype amino acid        sequence, and the one or more mutations improve the aqueous        solubility, stability, or resistance to chemical degradation of        the isolated peptide relative to a polypeptide comprising the        corresponding wildtype amino acid sequence. In certain        embodiments, the peptides according to the tenth aspect of the        invention also meet the structural features defining the        peptides according to the first or second aspect of the        invention.

A eleventh aspect of the invention relates to a fusion protein thatincludes one of the peptides of the first through eleventh aspects ofthe invention along with one or more of a purification tag, a solubilitytag, or a second peptide according to one of the first through tenthaspects of the invention.

A twelfth aspect of the invention relates to a composition that includesone or more peptides according to the first, second, third, fourth,fifth, sixth, seventh, eighth, ninth, or tenth aspects of the invention,or a fusion protein according to the eleventh aspect of the invention,and a carrier.

A thirteenth aspect of the invention relates to a method of impartingdisease resistance to plants. This method includes: applying aneffective amount of an isolated peptide according to the first, second,third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspects ofthe invention, a fusion protein according to the eleventh aspect of theinvention, or a composition according to the twelfth aspect of theinvention to a plant or plant seed or the locus where the plant isgrowing or is expected to grow, wherein said applying is effective toimpart disease resistance.

A fourteenth aspect of the invention relates to a method of enhancingplant growth. This method includes: applying an effective amount of anisolated peptide according to the first, second, third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth aspects of the invention, afusion protein according to the eleventh aspect of the invention, or acomposition according to the twelfth aspect of the invention to a plantor plant seed or the locus where the plant is growing or is expected togrow, wherein said applying is effective to enhance plant growth.

A fifteenth aspect of the invention relates to a method of increasing aplant's tolerance and resistance to biotic stressors. This methodincludes: applying an effective amount of an isolated peptide accordingto the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, or tenth aspects of the invention, a fusion protein according tothe eleventh aspect of the invention, or a composition according to thetwelfth aspect of the invention to a plant or plant seed or the locuswhere the plant is growing or is expected to grow, wherein said applyingis effective to increase the plant's tolerance and resistance to bioticstress factors selected from the group consisting of pests such asinsects, arachnids, nematodes, weeds, and combinations thereof.

A sixteenth aspect of the invention relates to a method of increasing aplant's tolerance to abiotic stress. This method includes: applying aneffective amount of an isolated peptide according to the first, second,third, fourth, fifth, sixth, seventh, eighth, ninth, or tenth aspects ofthe invention, a fusion protein according to the eleventh aspect of theinvention, or a composition according to the twelfth aspect of theinvention to a plant or plant seed or the locus where the plant isgrowing or is expected to grow, wherein said applying is effective toincrease the plant's tolerance to abiotic stress factors selected fromthe group consisting of salt stress, water stress (including drought andflooding), ozone stress, heavy metal stress, cold stress, heat stress,nutritional stress (phosphate, potassium, nitrogen deficiency),bleaching and light-induced stress, and combinations thereof.

A seventeenth aspect of the invention relates to a method impartingdesiccation resistance to cuttings removed from ornamental plants. Thismethod includes: applying an isolated peptide according to the first,second, third, fourth, fifth, sixth, seventh, eighth, ninth, or tenthaspects of the invention, a fusion protein according to the eleventhaspect of the invention, or a composition according to the twelfthaspect of the invention to a plant or the locus where the plant isgrowing, wherein said applying is effective to impart desiccationresistance to cuttings removed from the ornamental plant.

An eighteenth aspect of the invention relates to a method of impartingpost-harvest disease or post-harvest desiccation resistance to a fruitor vegetable. This method includes: applying an effective amount of anisolated peptide according to the first, second, third, fourth, fifth,sixth, seventh, eighth, ninth, or tenth aspects of the invention, afusion protein according to the eleventh aspect of the invention, or acomposition according to the twelfth aspect of the invention to a plantcontaining a fruit or vegetable or the locus where the plant is growing;or applying an effective amount of the isolated peptide or thecomposition to a harvested fruit or vegetable, wherein said applying iseffective to impart post-harvest disease resistance or desiccationresistance to the fruit or vegetable.

A nineteenth aspect of the invention relates to a method of enhancingthe longevity of fruit or vegetable ripeness. This method includes:applying an effective amount of an isolated peptide according to thefirst, second, third, fourth, fifth, sixth, seventh, eighth, ninth, ortenth aspects of the invention, a fusion protein according to theeleventh aspect of the invention, or a composition according to thetwelfth aspect of the invention to a plant containing a fruit orvegetable or the locus where the plant is growing; or applying aneffective amount of the isolated peptide or the composition to aharvested fruit or vegetable, wherein said applying is effective toenhance the longevity of fruit or vegetable ripeness.

A twentieth aspect of the invention relates to a method of modulatingone or more biological signaling processes of a plant. This methodincludes: applying an effective amount of an isolated peptide accordingto the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, or tenth aspects of the invention, a fusion protein according tothe eleventh aspect of the invention, or a composition according to thetwelfth aspect of the invention to a plant or the locus where the plantis growing, wherein said applying is effective in modulating one or morebiochemical signaling processes.

A twenty-first aspect of the invention relates to a DNA constructincluding a first nucleic acid molecule encoding a polypeptide accordingto the first, second, third, fourth, fifth, sixth, seventh, eighth,ninth, or tenth aspects of the invention or a fusion protein accordingto the eleventh aspect of the invention; and a promoter-effectivenucleic acid molecule operably coupled to the first nucleic acidmolecule. This aspect of the invention also encompasses a recombinantexpression vector containing the DNA construct, a recombinant host cellcontaining the DNA construct, as well as transgenic plants or plantseeds that include a recombinant plant cell of the invention (whichcontains the DNA construct).

A twenty-second aspect of the invention relates to a method of impartingdisease resistance to plants, enhance plant growth, impart tolerance andresistance to biotic stressors, impart tolerance to abiotic stress, ormodulating plant biochemical signaling. This method includes providing atransgenic plant transformed with a DNA construct according to thetwenty-first aspect of the invention; and growing the plant underconditions effective to permit the DNA construct to express the peptideor the fusion polypeptide to impart disease resistance, enhance plantgrowth, impart tolerance to biotic stress, impart tolerance to abioticstress, or modulate biochemical signaling to the transgenic plant.

A twenty-third aspect of the invention relates to a method of impartingdesiccation resistance to cuttings removed from ornamental plants,imparting post-harvest disease or post-harvest desiccation resistance toa fruit or vegetable, or enhancing the longevity of fruit or vegetableripeness. The method includes providing a transgenic plant transformedwith a DNA construct including a first nucleic acid molecule encoding apolypeptide according to the first, second, third, fourth, fifth, sixth,seventh, eighth, ninth, or tenth aspects of the invention or a fusionprotein according to the eleventh aspect of the invention; and growingthe plant under conditions effective to permit the DNA construct toexpress the peptide or the fusion polypeptide to impart desiccationresistance to cuttings removed from a transgenic ornamental plant,impart post-harvest disease resistance or desiccation resistance to afruit or vegetable removed from the transgenic plant, or enhancelongevity of ripeness for a fruit or vegetable removed from thetransgenic plant.

A twenty-fourth aspect of the invention relates to a method of impartingdisease resistance to plants, enhancing plant growth, impartingtolerance and resistance to biotic stressors, imparting tolerance toabiotic stress, or modulating biochemical signaling. This methodincludes providing a transgenic plant seed transformed with a DNAconstruct according to the twenty-first aspect of the invention;planting the transgenic plant seed in soil; and propagating a transgenicplant from the transgenic plant seed to permit the DNA construct toexpress the peptide or the fusion polypeptide to impart diseaseresistance, enhance plant growth, impart tolerance to biotic stress, orimpart tolerance to abiotic stress

A twenty-fifth aspect of the invention relates to a method of impartingdesiccation resistance to cuttings removed from ornamental plants,imparting post-harvest disease or post-harvest desiccation resistance toa fruit or vegetable, or enhancing the longevity of fruit or vegetableripeness. The method includes providing a transgenic plant seedtransformed with a DNA construct according to the twenty-first aspect ofthe invention; planting the transgenic plant seed in soil; andpropagating a transgenic plant from the transgenic plant seed to permitthe DNA construct to express the peptide or the fusion polypeptide toimpart desiccation resistance to cuttings removed from a transgenicornamental plant, impart post-harvest disease resistance or desiccationresistance to a fruit or vegetable removed from the transgenic plant, orenhance longevity of ripeness for a fruit or vegetable removed from thetransgenic plant.

By providing HR-eliciting peptides that exhibit improved solubility,stability, resistance to chemical degradation, or a combination of theseproperties, it will afford growers with greater flexibility inpreparing, handling, and delivering to plants in their fields orgreenhouses effective amounts of compositions containing theseHR-eliciting peptides. Simplifying the application process for growerswill lead to greater compliance and, thus, improved results with respectto one or more of disease resistance, growth enhancement, tolerance andresistance to biotic stressors, tolerance to abiotic stress, desiccationresistance for cuttings removed from ornamental plants, post-harvestdisease resistance or desiccation resistance to fruit or vegetablesharvested from plants, and/or improved longevity of fruit or vegetableripeness for fruit or vegetables harvested from plants. These and otherbenefits are described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a solubility and stability test of peptide P1 and P1mutants dissolved in deionized water. The following peptides are shown:P1 (SEQ ID NO: 4); P1-18A (SEQ ID NO: 44); P1-18K (SEQ ID NO: 45); andP1-18T (SEQ ID NO: 42). The curve for 1* is normalized to 100% of P1 atthe day 1 time point; 1** is the original P1 data.

FIG. 2 shows a solubility and stability test of peptide P1 and P1mutants dissolved in 50 mM citrate, pH 5.6. The following peptides areshown: P1, P1-18A, P1-18K, and P1-18T. The curve for 1* is normalized to100% of P1 at the day 1 time point; 1** is the original P1 data.

FIG. 3 shows a solubility and stability test of peptide P1 and P1mutants dissolved in 50 mM MES, pH 6. The following peptides are shown:P1, P1-18A, P1-18K, and P1-18T. The curve for 1* is normalized to 100%of P1 at the day 1 time point; 1** is the original P1 data.

FIG. 4 shows a solubility and stability test of peptide P1 and P1mutants dissolved in 50 mM MOPS, pH 6.5. The following peptides areshown: P1, P1-18A, P1-18K, and P1-18T.

FIG. 5 shows a solubility and stability test of peptide P1 and P1mutants dissolved in 50 mM citrate, pH 7.2. The following peptides areshown: P1, P1-18A, P1-18K, and P1-18T.

FIG. 6 shows a solubility and stability test of peptide P1 and P1mutants dissolved in 50 mM EDDS, pH 7.3. The following peptides areshown: P1, P1-18A, P1-18K, and P1-18T.

FIG. 7 shows a solubility and stability test of peptide P1 and P1mutants dissolved in 50 mM imidazole, pH 7.5. The following peptides areshown: P1, P1-18A, P1-18K, and P1-18T.

FIG. 8 shows a solubility and stability test of peptide P1 and P1mutants dissolved in 50 mM EDTA, pH 8. The following peptides are shown:P1, P1-18A, P1-18K, and P1-18T.

FIG. 9 shows a solubility and stability test of peptide P1 and P1mutants dissolved in phosphate, pH 8.0. The following peptides areshown: P1, P1-18A, P1-18K, and P1-18T.

FIG. 10 shows a solubility and stability test of peptide P1 and P1mutants dissolved in 50 mM TES, pH 8.0. The following peptides areshown: P1, P1-18A, P1-18K, and P1-18T.

FIG. 11 shows a solubility and stability test of peptide P4 and P4mutants dissolved in deionized water. The following peptides are shown:P1, P1-18A, P1-18K, and P1-18T.

FIG. 12 shows a solubility and stability test of peptide P4 and P4mutants dissolved in 50 mM citrate, pH 5.6. The following peptides areshown: P4 (SEQ ID NO: 5); P4-14A (SEQ ID NO: 136); P4-14D (SEQ ID NO:137); P4-14K (SEQ ID NO: 138); P4-14Q (SEQ ID NO: 139); and P4-14S (SEQID NO: 6).

FIG. 13 shows a solubility and stability test of peptide P4 and P4mutants dissolved in 50 mM MES, pH 6. The following peptides are shown:P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

FIG. 14 shows a solubility and stability test of peptide P4 and P4mutants dissolved in 50 mM MOPS, pH 6.5. The following peptides areshown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

FIG. 15 shows a solubility and stability test of peptide P4 and P4mutants dissolved in 50 mM citrate, pH 7.2. The following peptides areshown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

FIG. 16 shows a solubility and stability test of peptide P4 and P4mutants dissolved in 50 mM EDDS, pH 7.3. The following peptides areshown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

FIG. 17 shows a solubility and stability test of peptide P4 and P4mutants dissolved in 50 mM imidazole, pH 7.5. The following peptides areshown: P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

FIG. 18 shows a solubility and stability test of peptide P4 and P4mutants dissolved in 50 mM EDTA, pH 8. The following peptides are shown:P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

FIG. 19 shows a solubility and stability test of peptide P4 and P4mutants dissolved in phosphate, pH 8. The following peptides are shown:P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

FIG. 20 shows a solubility and stability test of peptide P4 and P4mutants dissolved in 50 mM TES, pH 8. The following peptides are shown:P4, P4-14A, P4-14D, P4-14K, P4-14Q, and P4-14S.

FIG. 21 shows a comparison of the stability of peptide P4 and P4 mutantsdissolved in 30% isopropanol, 5 mM DTPA, 0.5% sodium thiosulfate, and 50mM TES pH 8. The following peptides are shown: P4, P4-14A, P4-14D,P4-14K, P4-14Q, and P4-14S

FIG. 22 shows a comparison of the stability of various peptidesdissolved in 50 mM TES pH 8. The following peptides are shown: P1 (SEQID NO: 4); P4-14S (SEQ ID NO: 6); P1-1S (SEQ ID NO: 109); P1-14S (SEQ IDNO: 110); P1-18Q (SEQ ID NO: 115); and P1-23P (SEQ ID NO:118).

FIG. 23 shows a solubility and stability test of peptide P18 (SEQ ID NO:83) dissolved in either MES pH 6, MOPS pH 6.5, EDDS pH 7.3, imidazole pH7.5, or EDTA pH 8.

FIG. 24 shows a solubility and stability test of peptide P18 and P18mutants dissolved in 50 mM EDTA, pH 8. The following peptides are shown:P18 (SEQ ID NO: 83), P18-1 (SEQ ID NO: 163), and P18-4 (SEQ ID NO: 164).

FIG. 25 shows a stability test of peptide P19 (SEQ ID NO: 89) andP19-20L (SEQ ID NO: 90) mutant dissolved in 50 mM citrate, pH 7.2.

FIG. 26 shows a stability test of peptide P19 (SEQ ID NO: 89) andP19-20L (SEQ ID NO: 90) mutant dissolved in 50 mM TES, pH 8.0.

DETAILED DESCRIPTION OF THE INVENTION

One aspect of the invention relates to novel peptides that possess theability to induce a hypersensitive response in plants and promote activeplant responses that afford one or more of the following attributes:disease resistance, growth enhancement, tolerance and resistance tobiotic stressors, tolerance to abiotic stress, desiccation resistancefor cuttings removed from ornamental plants, post-harvest diseaseresistance or desiccation resistance to fruit or vegetables harvestedfrom plants, and/or improved longevity of fruit or vegetable ripenessfor fruit or vegetables harvested from plants.

As used herein, naturally occurring amino acids are identifiedthroughout by the conventional three-letter and/or one-letterabbreviations, corresponding to the trivial name of the amino acid, inaccordance with the following list: Alanine (Ala, A), Arginine (Arg, R),Asparagine (Asn, N), Aspartic acid (Asp, D), Cysteine (Cys, C), Glutamicacid (Glu, E), Glutamine (Gln, Q), Glycine (Gly, G), Histidine (His, H),Isoleucine (Ile, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met,M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine(Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Val, V).The abbreviations are accepted in the peptide art and are recommended bythe IUPAC-IUB commission in biochemical nomenclature. Naturallyoccurring variations of amino acids include, without limitation,gamma-glutamate (g-Glu) and isoaspartate (iso-Asp or isoD).

The term “amino acid” further includes analogues, derivatives, andcongeners of any specific amino acid referred to herein, as well asC-terminal or N-terminal protected amino acid derivatives (e.g.,modified with an N-terminal, C-terminal, or side-chain protecting group,including but not limited to acetylation, formylation, methylation,amidation, esterification, PEGylation, and addition of lipids.Non-naturally occurring amino acids are well known and can be introducedinto peptides of the present invention using solid phase synthesis asdescribed below. Furthermore, the term “amino acid” includes both D- andL-amino acids. Hence, an amino acid which is identified herein by itsname, three letter or one letter symbol and is not identifiedspecifically as having the D or L configuration, is understood to assumeany one of the D or L configurations. In one embodiment, a peptidecomprises all L-amino acids.

In certain embodiments, peptides are identified to “consist of” arecited sequence, in which case the peptide includes only the recitedamino acid sequence(s) without any extraneous amino acids at the N- orC-terminal ends thereof. To the extent that a recited sequence is in theform of a consensus sequence where one or more of the denoted X or Xaaresidues can be any of one or more amino acids, then multiple peptidesequences are embraced by a peptide consisting of such a recitedsequence.

In certain other embodiments, peptides are identified to “consistessentially of” a recited sequence, in which case the peptide includesthe recited amino acid sequence(s) optionally with one or moreextraneous amino acids at the N- and/or C-terminal ends thereof, whichextraneous amino acids do not materially alter one or more of thefollowing properties: (i) the ability of the peptide to induce ahypersensitive response in plants, (ii) solubility of the peptide inwater or aqueous solutions, (iii) stability of the peptide dissolved inwater or aqueous solution at 50° C. over a period of time (e.g., 3weeks), and (iv) resistance of the peptide to chemical degradation inthe presence of an aqueous buffered solution that includes a biocidalagent (e.g., Proxel®GXL) at 50° C. over a period of time (e.g., 3weeks).

Briefly, the stability and resistance to chemical degradation ofpeptides can be assessed as follows using peptide samples having aninitial purity of at least about 80%, at least about 82%, at least about84%, at least about 86%, at least about 88%, at least about 90%, atleast about 92%, at least about 94%, at least about 96%, or at leastabout 98%. For water stability, the peptide is dissolved directly inde-ionized water. For chemical degradation tests, the peptide isdissolved in an aqueous solution containing 50 mM pH buffer and 0.25%Proxel GXL. Exemplary pH buffers include, without limitation: (i)Citrate pH 5.6; (ii) MES pH 6.2; (iii) MOPS pH 6.5; (iv) imidazole pH7.0; (v) Citrate pH 7.2; (vi) EDDS, pH 7.3; (vii) EDTA pH 8.0; (viii)sodium phosphate pH 8.0; or (ix) TES pH 8.0. Peptides are firstdissolved in the aqueous solution at a concentration of 0.5 mg/ml. Thesamples are incubated at 50° C. to allow for accelerated degradation. Aninitial sample of the peptide is removed, diluted 10× with water, andanalyzed by reverse-phase HPLC. Briefly, 20 μl of the sample is injectedinto the solvent flow of an HPLC instrument and analyzed on a C18 HPLCcolumn (YMC ProPack C18, YMC, Japan, or C18 Stablebond, AgilentTechnologies, USA) using either a triethylamine phosphate inwater/acetonitrile gradient or a 0.1% TFA in water/0.1% TFA inacetonitrile gradient to separate different peptide species. Elutingpeptides are monitored by UV absorbance at 218 nm and quantified basedon the area under the peak. The area under the peak for the initialpeptide sample is treated as the standard for relative quantification insubsequent runs. At regular intervals (e.g., 1, 3, 7, 10, 14, 17, and 21days), each peptide sample is surveyed and analyzed by HPLC as describedabove. If necessary to observe degradation (i.e., where the peptideexhibits a high degree of chemical stability), this protocol can beextended by several weeks to observe degradation. The quantification ofsubsequent peptide runs is expressed as a percentage of the original(day 0) HPLC result.

A peptide that is at least partially soluble in water or aqueoussolution exhibits a solubility of greater than 0.1 mg/ml, preferably atleast about 1.0 mg/ml, at least about 2.0 mg/ml, at least about 3.0mg/ml, or at least about 4.0 mg/ml. In certain embodiments, the peptideexhibits high solubility in water or aqueous solution, with a solubilityof at least about 5.0 mg/ml, at least about 10.0 mg/ml, at least about15.0 mg/ml, or at least about 20 mg/ml.

A peptide that is stable in water or aqueous solution exhibits at leastabout 66%, at least about 68%, at least about 70%, at least about 72%,at least about 74%, at least about 76%, at least about 78%, at leastabout 80%, at least about 82%, at least about 84%, at least about 86%,at least about 88%, or at least about 90% of the original peptideconcentration over the designated period of time incubated at 50° C. Incertain embodiments, the designated period of time is 3 days, 7 days, 14days, 21 days, 28 days, one month, two months, or three months.

A peptide that is resistant to chemical degradation exhibits at leastabout 66%, at least about 68%, at least about 70%, at least about 72%,at least about 74%, at least about 76%, at least about 78%, at leastabout 80%, at least about 82%, at least about 84%, at least about 86%,at least about 88%, or at least about 90% of the original peptideconcentration over the designated period of time incubated at 50° C. Incertain embodiments, the designated period of time is 3 days, 7 days, 14days, 21 days, 28 days, one month, two months, or three months.

A property of a peptide to elicit a hypersensitive response, or not,upon infiltration or application of the peptide to plant tissues can bemeasured by applying the peptide in dry powder form or in solution formto a plant, particularly though not exclusively a plant leaf.Application rates include 1-500 ug/ml for liquid solution and0.0001-0.5% (w/w for powder application. Exemplary application of thepeptide in solution form is described in the accompanying Examples.Plants are considered HR-positive (“HR+”) if they exhibit wide-spreadmacroscopic cell death visible to the naked eye, accompanied by wiltingand browning of the affected tissue within 48 hours. Plants areconsidered HR-negative (“HR−”) if they exhibit no discernible wilting ortissue death observable by naked eye.

In certain embodiments, material alteration of the one or moreproperties is intended to mean that there is less than 20% variation,less than 15% variation, less than 10% variation, or less than 5%variation in a recited property when comparing a peptide possessing theone or more extraneous amino acids to an otherwise identical peptidelacking the one or more extraneous amino acids. In certain embodiments,the number of extraneous amino acids at the N- or C-terminal ends is upto 20 amino acids at one or both ends, up to 15 amino acids at one orboth ends, up to 10 amino acids at one or both ends, up to 7 amino acidsat one or both ends, up to 5 amino acids at one or both ends, or up to 3amino acids at one or both ends. Further, to the extent that a recitedsequence is in the form of a consensus sequence where one or more of thedenoted X or Xaa residues can be any of one or more amino acids, thenmultiple peptide sequences are embraced by the peptide consistingessentially of such a recited sequence, without regard to additionalvariations of such sequences that are afforded by the presence ofextraneous amino acids at the N- and/or C-terminal ends thereof.

In various embodiments of the invention, the disclosed peptides mayinclude a hydrophilic amino acid sequence, e.g., at either theN-terminal or C-terminal end of a designated peptide sequence. Thehydrophilic amino acid sequence is at least 3, at least 4, at least 5,at least 6, at least 7, at least 8, at least 9, or at least 10 aminoacids in length, and includes amino acid residues that contribute to ahydrophilic property of the amino acid sequence that is adjacent to theamino acid sequence of the designated peptide (i.e., the peptide thatinduces an active plant response).

Different methods have been used in the art to calculate the relativehydrophobicity/hydrophilicity of amino acid residues and proteins (Kyteet al., “A Simple Method for Displaying the Hydropathic Character of aProtein,” J. Mol. Biol. 157: 105-32 (1982); Eisenberg D,“Three-dimensional Structure of Membrane and Surface Proteins,” Ann.Rev. Biochem. 53: 595-623 (1984); Rose et al., “Hydrogen Bonding,Hydrophobicity, Packing, and Protein Folding,” Annu. Rev. Biomol.Struct. 22: 381-415 (1993); Kauzmann, “Some Factors in theInterpretation of Protein Denaturation,” Adv. Protein Chem. 14: 1-63(1959), which are hereby incorporated by reference in their entirety).Any one of these hydrophobicity scales can be used for the purposes ofthe present invention; however, the Kyte-Doolittle hydrophobicity scaleis perhaps the most often referenced scale. These hydropathy scalesprovide a ranking list for the relative hydrophobicity of amino acidresidues. For example, amino acids that contribute to hydrophilicityinclude Arg (R), Lys (K), Asp (D), Glu (E), Gln (Q), Asn (N), and His(H) as well as, albeit to a lesser extent, Ser (S), Thr (T), Gly (G),Pro (P), Tyr (Y), and Trp (W). For example, polyglutamate sequences canbe used to enhance solubility of proteins and other drug molecules(Lilie et al, Biological Chemistry 394(8):995-1004(2013); Li et al.,Cancer Research 58: 2404-2409(1998)), each of which is herebyincorporated by reference in its entirety).

The “hydropathy index” of a protein or amino acid sequence is a numberrepresenting its average hydrophilic or hydrophobic properties. Anegative hydropathy index defines the hydrophilicity of the amino acidsequence of interest. The hydropathy index is directly proportional tothe hydrophilicity of the amino acid sequence of interest; thus, themore negative the index, the greater its hydrophilicity. In certainembodiments, the added hydrophilic amino acid sequence described abovehas a hydropathy index of less than 0, −0.4, −0.9, −1.3, −1.6, −3.5,−3.9, or −4.5. In certain embodiments, the resulting entire peptide willhave a hydropathy index of less than 0.3, 0.2, 0.1, or 0.0, preferablyless than −0.1, −0.2, −0.3, −0.4, more preferably less than −0.5, −0.6,−0.7, −0.8, −0.9, or −1.0.

In the peptides of the present invention, amino acids that contribute toa hydrophilic hydropathy index, for either the peptide as a whole or theadded hydrophilic amino acid sequence, include Arg (R), Lys (K), Asp(D), Glu (E), Gln (Q), Asn (N), His (H), Ser (S), Thr (T), Gly (G), Pro(P), Tyr (Y), and Trp (W). Of these, Asp (D), Glu (E), Gln (Q), Asn (N)or their variants are preferred. Exemplary variants include g-glutamatefor Glu and isoaspartic acid (or isoD) for Asp.

As used herein, in this and in other aspects of the invention, the term“hydrophobic amino acid” is intended to refer to an amino acid thatcontributes hydrophobicity to the hydropathy index of a designated aminoacid sequence. Amino acids that contribute to a hydrophobic hydropathyindex, for either the peptide as a whole or a particular amino acidsequence thereof, include Ile (I), Val (V), Leu (L), Phe (F), Cys (C),Met (M), and Ala (A). In certain embodiments, the term “hydrophobicamino acid” may refer to any one of Ile (I), Val (V), Leu (L), Phe (F),Cys (C), Met (M), and Ala (A); or, alternatively, to any one of Ile (I),Val (V), Leu (L), Phe (F), and Ala (A). In certain other embodiments,the term “hydrophobic amino acid” may refer to one of Ile (I), Val (V),Leu (L), and Phe (F).

As used herein, the term “non-hydrophobic amino acid” is intended tomean an amino acid that is hydrophilic (or not hydrophobic) on one ofthe above-identified hydrophobicity scales. This term generally refersto those amino acids that contribute to a hydrophilic hydropathy indexfor either the peptide as a whole or the added hydrophilic amino acidsequence.

In one aspect of the invention, the peptide includes the amino acidsequence of:(L/I/V/F)-X—X-(L/I/V/F)-(L/I)-X—X-(L/I/V/F)-(L/I/V/A)-X—X-(L/I)-(L/I/V/F)(SEQ ID NO: 93) wherein the peptide is free of cysteine and methionine;each X at positions 2 and 6 is optional and, when present, is any aminoacid, including any naturally occurring amino acid; and each X atpositions 3, 7, 10, and 11 is any amino acid, including any naturallyoccurring amino acid.

In a related aspect of the invention, the peptide includes the aminoacid sequence of SEQ ID NO: 93 (shown above), wherein the peptide isfree of cysteine and methionine; each X at positions 2, 6, and 10 isoptional and, when present, is any amino acid, including any naturallyoccurring amino acid; and each X at positions 3, 7, and 11 is any aminoacid, including any naturally occurring amino acid.

According to one embodiment, one or more of X at positions 2, 6, and 10is not present (i.e., the gap between the first hydrophobic amino acidand the first hydrophobic amino acid doublet is reduced from two to oneamino acid residue and/or the gap between the first and secondhydrophobic amino acid doublets is reduced from two to one amino acidresidue and/or the gap between the second and third hydrophobic aminoacid doublets is reduced from two to one amino acid residue). In thisembodiment, it is contemplated that these peptides exclude the aminoacid at only one of positions 2, 6, and 10.

In an alternative embodiment, X at both of positions 2 and 6 is present(i.e., the gap between the hydrophobic amino acids is maintained at twoamino acid residues at both locations).

In another embodiment, X at each of positions 2, 6, and 10 is present(i.e., the gap between the hydrophobic amino acids is maintained at twoamino acid residues at each location).

In certain embodiments, SEQ ID NO: 93 may further include an additionalamino acid residue between the hydrophobic doublets (two of L/I/V/F/A,as indicated) and the additional amino acid can be any amino acid. Inthese embodiments, the gap before the first hydrophobic doublet is threeamino acids, the gap between the first and second hydrophobic doubletsis three amino acids, the gap between the second and third hydrophobicdoublet is three amino acids, or combinations thereof.

The peptide length in this embodiment is less than 100 amino acids, oralternatively less than 90 amino acids, less than 80 amino acids, lessthan 70 amino acids, less than 60 amino acids, or less than about 50amino acids. In certain embodiments, the peptide length is between 13and about 50 amino acids in length.

In the embodiments described above, where X at each of positions 2, 3,6, 7, 10, and 11 (when present) of SEQ ID NO: 93 can be any amino acid,in certain embodiments these residues are hydrophilic in nature. Asdescribed above, these hydrophilic amino acids include Arg (R), Lys (K),Asp (D), Glu (E), Gln (Q), Asn (N), His (H), Ser (S), Thr (T), Gly (G),Pro (P), Tyr (Y), and Trp (W). Of these, Asp (D), Glu (E), Gln (Q), Asn(N) or their variants are preferred. Exemplary variants includeg-glutamate for Glu and isoaspartic acid (or isoD) for Asp.

In this embodiment, the isolated peptide is stable when dissolved inwater; resistant to chemical degradation in aqueous conditions in thepresence of a pH buffer and a biocide, as described above; and/or has asolubility in an aqueous solution of at least about 1.0 mg/ml.

Another aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

XXGISEKXXXXXXXXXXXXXXXX (SEQ ID NO: 1, P1/P4 consensus), wherein

-   X at position 1 is optional and can be S, N, D, isoD, G, A, or S;-   X at position 2 is optional and can be Q, E, g-glutamate, G, A, or    S;-   X at position 8 is Q, E, g-glutamate, G, A, or S;-   X at position 9 is L, I, F, or V;-   X at position 10 is optional and can be D or isoD;-   X at position 11 is Q, E, g-glutamate, G, A, or S;-   X at position 12 is M, L, I, or F;-   X at position 13 is M, L, or I;-   X at position 14 is optional and can be any hydrophilic amino acid,    preferably C, S, T, A, D, isoD, K, or Q;-   X at position 15 is Q, E, g-glutamate, G, A, S, K, or I;-   X at position 16 is M, L, I, V, or F;-   X at position 17 is M, L, I, A, or V;-   X at position 18 is Q, E, g-glutamate, G, A, S, M, T, or K;-   X at position 19 is A, D, isoD, S, V, T, K, R, E, g-glutamate, H, or    G;-   X at position 20 is M, L, or I;-   X at position 21 is M, L, I, V, S, or F;-   X at position 22 is Q, E, g-glutamate, G, A, S;-   X at position 23 is P, Q, E, g-glutamate, G, A, or S; and    wherein the isolated peptide comprises one or more mutations    relative to a corresponding wildtype amino acid sequence. In certain    embodiments, the one or more mutations improve the aqueous    solubility, stability, or resistance to chemical degradation of the    isolated peptide relative to a polypeptide comprising the    corresponding wildtype amino acid sequence.

In certain embodiments, these peptides according to the second aspect ofthe invention also meet the structural features defining the peptides ofSEQ ID NO: 93, in which case methionine and cysteine residues are notpresent.

In this embodiment, the corresponding wildtype amino acid sequence, forpurposes of comparing properties of the inventive peptide, is apolypeptide comprising or the peptide consisting of the amino acidsequence of NQGISEKQLDQLLTQLIMALLQQ (P1, SEQ ID NO: 4) orSQGISEKQLDQLLCQLIQALL (amino acids 1-21 of SEQ ID NO: 5, P4). P1 (SEQ IDNO: 4) is derived from the full length protein of Xanthomonas harpinHpaG (Kim et al., “Mutational Analysis of Xanthomonas Harpin HpaGIdentifies a Key Functional Region That Elicits the HypersensitiveResponse in Nonhost Plants,” J. Bacteriol. 186(18):6239-6247 (2004),which is hereby incorporated by reference in its entirety). P4 (SEQ IDNO: 5) is derived from the full length harpin of Xanthomonas oryzae pv.oryzae (Ji et al., “Two Coiled-Coil Regions of Xanthomonas oryzae pv.Oryzae Harpin Differ in Oligomerization and Hypersensitive ResponseInduction,” Amino Acids 40:381-392 (2011), which is hereby incorporatedby reference in its entirety).

In this embodiment, the isolated peptide is stable when dissolved inwater; resistant to chemical degradation in aqueous conditions in thepresence of a pH buffer and a biocide, as described above; and/or has asolubility in an aqueous solution of at least about 1.0 mg/ml.

The length of peptides according to this second aspect is preferablyless than about 100 amino acids, or alternatively less than 90 aminoacids, less than 80 amino acids, less than 70 amino acids, less than 60amino acids, or less than about 50 amino acids. In certain embodiments,the peptide length is between 23 and about 50 amino acids in length.

One exemplary family of peptides according to the second aspect of theinvention have the amino acid sequence of:

SXGISEKXXDXXXXXXXXAXXXP (SEQ ID NO: 2, P4 consensus), wherein

-   X at position 2 is Q, E, g-glutamate, G, A, or S;-   X at position 8 is Q, E, g-glutamate, G, A, or S;-   X at position 9 is L, A, D, isoD, I, V, or F;-   X at position 11 is Q, E, g-glutamate, G, A, or S;-   X at position 12 is L, D, isoD, I, or F;-   X at position 13 is L, I, V, or F;-   X at position 14 is any hydrophilic amino acid, preferably C, S, or    T, S or T, or only S;-   X at position 15 is Q, E, g-glutamate, G, A, S, K, or I-   X at position 16 is L, A, I, V, M, or F-   X at position 17 is I, S, or F-   X at position 18 is Q, E, g-glutamate, G, A, or S;-   X at position 20 is L, I, V, or F;-   X at position 21 is L or F; and-   X at position 22 is Q, E, g-glutamate, G, A, or S.

In certain embodiments, these peptides according to SEQ ID NO: 2 alsomeet the structural features defining the peptides of SEQ ID NO: 93, inwhich case methionine and cysteine residues are not present. Thus, inthese embodiments, X at position 14 is S or T, preferably S.

Exemplary peptides that share the consensus structure with SEQ ID NO: 2,or are derived from SEQ ID NO: 2 and meet the consensus structure of SEQID NO: 93, are identified in Table 1 below:

TABLE 1 Peptide Variants of Peptide P4 (SEQ ID NO: 5) SEQ Peptide IDName Sequence NO: P4 SQGISEKQLDQLLCQLIQALLQP 5 P4-14SSQGISEKQLDQLLSQLIQALLQP 6 P4-14s-18e SEGISEKQLDQLLSQLIEALLQP 191P4-14s-18s SQGISEKQLDQLLSQLISALLQP 7 P4-14s-2,18eSEGISEKQLDQLLSQLIEALLQP 8 P4-2e-8e SEGISEKELDQLLSQLIQALLQP 9P4-2e-8e-15e SEGISEKELDQLLSELIQALLQP 10 P4-allE SEGISEKELDELLSELIEALLQP11 P4-14s-9i SQGISEKQIDQLLSQLIQALLQP 196 P4-14s-9vSQGISEKQVDQLLSQLIQALLQP 19 P4-14s-9f SQGISEKQFDQLLSQLIQALLQP 20P4-14s-12i SQGISEKQLDQILSQLIQALLQP 22 P4-14s-12f SQGISEKQLDQFLSQLIQALLQP23 P4-14s-13i SQGISEKQLDQLISQLIQALLQP 24 P4-14s-15aSQGISEKQLDQLLSALIQALLQP 27 P4-14s-15k SQGISEKQLDQLLSKLIQALLQP 28P4-14s-15s SQGISEKQLDQLLSSLIQALLQP 29 P4-14s-15i SQGISEKQLDQLLSILIQALLQP30 P4-14s-16i SQGISEKQLDQLLSQIIQALLQP 32 P4-14s-16fSQGISEKQLDQLLSQFIQALLQP 34 P4-14S-20i SQGISEKQLDQLLSQLIQAILQP 37P4-14s-21f SQGISEKQLDQLLSQLIQALFQP 40 P4-14s-dN6       KQLDQLLSQLIQALLQP94 P4-14s-dN5      EKQLDQLLSQLIQALLQP 95 P4-14s-dN4    SEKQLDQLLSQLIQALLQP 96 P4-14s-dN2   GISEKQLDQLLSQLIQALLQP 97P4-14s-3E SQEISEKQLDQLLSQLIQALLQP 98 P4-14S-4L SQGLSEKQLDQLLSQLIQALLQP99 P4-14S-4A SQGASEKQLDQLLSQLIQALLQP 100 P4-14S-4DSQGDSEKQLDQLLSQLIQALLQP 101 P4-14s-5V SQGIVEKQLDQLLSQLIQALLQP 102P4-14s-6R SQGISRKQLDQLLSQLIQALLQP 103 P4-14s-6V SQGISVKQLDQLLSQLIQALLQP104 P4-14s-7D SQGISEDQLDQLLSQLIQALLQP 105 P4-14S-7VSQGISEVQLDQLLSQLIQALLQP 106 P4-14S-8V SQGISEKVLDQLLSQLIQALLQP 107P4-14S-8S SQGISEKSLDQLLSQLIQALLQP 108 P4-14S-10V SQGISEKQLVQLLSQLIQALLQP111 P4-14S-11V SQGISEKQLDVLLSQLIQALLQP 112 P4-14S-12V SQGISEKQLDQVLSQLIQALLQP 113 P4-14S-12S  SQGISEKQLDQSLSQLIQALLQP 114P4-14S-14V  SQGISEKQLDQLLVQLIQALLQP 116 P4-14S-15V SQGISEKQLDQLLSVLIQALLQP 117 P4 SQGISEKQLDQLLCQLIQALLQP 5 P4-14s-17LSQGISEKQLDQLLSQLLQALLQP 119 P4-14S-17A SQGISEKQLDQLLSQLAQALLQP 120P4-14s-17V SQGISEKQLDQLLSQLVQALLQP 121 P4-14S-18VSQGISEKQLDQLLSQLIVALLQP 122 P4-14S-19V SQGISEKQLDQLLSQLIQVLLQP 123P4-14S-19D SQGISEKQLDQLLSQLIQDLLQP 124 P4-14S-19SSQGISEKQLDQLLSQLIQSLLQP 125 P4-14S-21S SQGISEKQLDQLLSQLIQALSQP 127P4-14S-21I SQGISEKQLDQLLSQLIQALIQP 128 P4-14S-21VSQGISEKQLDQLLSQLIQALVQP 129 P4-14S-22V SQGISEKQLDQLLSQLIQALLVP 130P4-14S-dC2 SQGISEKQLDQLLSQLIQALL 131 p4-d10 SQGISEKQL_QLLSQLIQALLQP 132p4-d14 SQGISEKQLDQLL_QLIQALLQP 133 P4-14A SQGISEKQLDQLLAQLIQALLQP 136p4-14D SQGISEKQLDQLLDQLIQALLQP 137 P4-14K SQGISEKQLDQLLKQLIQALLQP 138P4-14Q SQGISEKQLDQLLQQLIQALLQP 139 p4-i9A SQGISEKQALDQLLSQLIQALLQP 140polyE-minp4    SEEEEELDQLLSQLIQALLQP 232 polyE-min2p4   SEEEEELDQLLSQLIQALLQ 31 polyE-min3P4    SEEEEELDQLLSQLIQALL 33P4-NpolyR   RRRRRGGLDQLLSQLIQALLQP 35 P4-CpolyR         LDQLLSQLIQALLQPGGRRRRR 36 P4-NpolyK   KKKKKGGLDQLLSQLIQALLQP 38P4-CpolyK          LDQLLSQLIQALLQPGGKKKKK 39 P4-7E-cRSQGISEEQLDQLLSQLIQALLQPR 104 minp4-PolyE          LDQLLSQLIQALLEEEEE 192SEE-minp4-EE       SEELDQLLSQLIQALLEE 193Select peptides in Table 1 include solubility tags, indicated by italicprint, including SEEEEE, SEE, EEEEE, EE, RRRRRGG, KKKKKGG, GGKKKKK, andGGRRRRR. Peptides comprising the sequences shown in Table 1 but lackingthese specific solubility tags (or having a different solubility tag)are also contemplated herein.

As noted above, the peptide P4 (SEQ ID NO: 5) is derived from the harpinof Xanthomonas oryzae pv. oryzae (Ji et al., “Two Coiled-Coil Regions ofXanthomonas oryzae pv. Oryzae Harpin Differ in Oligomerization andHypersensitive Response Induction,” Amino Acids 40:381-392 (2011), whichis hereby incorporated by reference in its entirety). Ji et al.discloses a fragment of this harpin having the amino acid sequenceSQGISEKQLDQLLCQLIQALL (i.e., amino acids 1-21 of SEQ ID NO: 5). Incertain embodiments, an isolated peptide comprising the amino acidsequence of SEQ ID NO: 5 is a peptide that has an overall length of lessthan about 100 amino acids (i.e., from 23 amino acids up to about 100amino acids in length). In certain other embodiments, the isolatedpeptide consists essentially of SEQ ID NO: 5, whereas in anotherembodiment the isolated peptide consists of SEQ ID NO: 5.

Another exemplary family of peptides according to the second aspect ofthe invention have the amino acid sequence of:

XXGISEKXLDXLLTXLIXALLXX (SEQ ID NO: 3, P1 consensus), wherein

-   X at position 1 is N, D, isoD, G, A, or S;-   X at position 2 is Q, E, g-glutamate, G, A, or S;-   X at position 8 is Q, E, g-glutamate, G, A, or S;-   X at position 11 is Q, E, g-glutamate, G, A, or S;-   X at position 15 is Q, E, g-glutamate, G, A, or S;-   X at position 18 is M, T, K, E, g-glutamate, G, A, or S;-   X at position 22 is Q, E, g-glutamate, G, A, or S; and-   X at position 23 is Q, E, g-glutamate, G, A, or S.

In certain embodiments, these peptides according to SEQ ID NO: 3 alsomeet the structural features defining the peptides of SEQ ID NO: 93, inwhich case methionine and cysteine residues are not present. Thus, inthose embodiments, X at position 18 is T, K, E, g-glutamate, G, A, or S.

In certain embodiments, the peptides sharing the structure of SEQ ID NO:3 have at least one of the residues at positions 2, 8, 11, 15, 22, and23 of SEQ ID NO: 3 being other than Gln (Q), i.e., being E, g-glutamate,G, A, or S. In certain embodiments, two or more of the residues atpositions 2, 8, 11, 15, 22, and 23 of SEQ ID NO: 3 are other than Gln(Q), including three, four, five, or all six of these residues beingother than Gln (Q).

Exemplary peptides that share the consensus structure with SEQ ID NO: 3,or are derived from SEQ ID NO: 3 and meet the consensus structure of SEQID NO: 93, are identified in Table 2 below:

TABLE 2 Peptide Variants of Peptide P1 (SEQ ID NO: 4) SEQ Peptide IDName Sequence NO: P1 NQGISEKQLDQLLTQLIMALLQQ 4 P1-2ENEGISEKQLDQLLTQLIMALLQQ 41 P1-18T NQGISEKQLDQLLTQLITALLQQ 42 P1-18ENQGISEKQLDQLLTQLIEALLQQ 43 P1-18A NQGISEKQLDQLLTQLIAALLQQ 44 P1-18KNQGISEKQLDQLLTQLIKALLQQ 45 P1-2E,8E,11E, NEGISEKELDELLTELIEALLQQ 4615E,18E P1-1S SQGISEKQLDQLLTQLIMALLQQ 109 P1-14S NQGISEKQLDQLLSQLIMALLQQ110 P1-18Q NQGISEKQLDQLLTQLIQALLQQ 115 P1-23P NQGISEKQLDQLLTQLIMALLQP118 polyE-minP1   SEEEEELDQLLTQLIEALLQQ 126 polyE-min2P1  SEEEEELDQLLTQLIEALLQ 134 polyE-min3P1   SEEEEELDQLLTQLIEALL 141p1-allE-17A NEGISEKELDELLTELAEALLQQ 195Select peptides in Table 2 include solubility tags, indicated by italicprint, including SEEEEE. Peptides comprising the sequences shown inTable 2 but lacking this specific solubility tag (or having a differentsolubility tag) are also contemplated herein.

As noted above, the peptide of SEQ ID NO: 4 is derived from the harpinof Xanthomonas oryzae pv. oryzae (Ji et al., “Two Coiled-Coil Regions ofXanthomonas oryzae pv. Oryzae Harpin Differ in Oligomerization andHypersensitive Response Induction,” Amino Acids 40:381-392 (2011), whichis hereby incorporated by reference in its entirety).

Yet another aspect of the invention relates to an isolated peptidehaving the amino acid sequence of:

-   (i) KPXDSXSXIAKLISXLIXSLLX (SEQ ID NO: 47, P15b/P20 consensus),    wherein    -   X at position 3 is N, D, or isoD;    -   X at position 6 is Q, E, g-glutamate, G, A, or S;    -   X at position 8 is N, D, or isoD;    -   X at position 15 is optional and can be any amino acid;    -   X at position 18 is M, E, g-glutamate, G, A, S, T, or K; and    -   X at position 22 is optional and can be Q, E, g-glutamate, G, A,        or S; or-   (ii) IAKLISXLIXSLLX (SEQ ID NO: 12, P15/20 min consensus), wherein    -   X at position 7 is optional and can be any amino acid;    -   X at position 10 is M, E, g-glutamate, G, A, S, T, or K; and    -   X at position 14 is optional and can be Q, E, g-glutamate, G, A,        or S.

In certain embodiments, these peptides according to SEQ ID NO: 47 or 12also meet the structural features defining the peptides of SEQ ID NO:93, in which case methionine and cysteine residues are not present.Thus, in those embodiments, X at position 15 of SEQ ID NO: 47 is otherthan M, and X at position 18 of SEQ ID NO: 47 is E, g-glutamate, G, A,S, T, or K. Similarly, X at position 7 of SEQ ID NO: 12 is other than M,and X at position 10 of SEQ ID NO: 47 is E, g-glutamate, G, A, S, T, orK.

The length of peptides according to this third aspect is preferably lessthan about 100 amino acids, or alternatively less than 90 amino acids,less than 80 amino acids, less than 70 amino acids, less than 60 aminoacids, or less than about 50 amino acids. In certain embodiments, thepeptide is between 20 and 44 amino acids in length.

In certain embodiments, the peptides sharing the structure of SEQ ID NO:47 have at least one of the residues at positions 6 and 22 of SEQ ID NO:47 being other than Gln (Q), i.e., being E, g-glutamate, G, A, or S. Incertain embodiments, both the residues at positions 6 and 22 of SEQ IDNO: 47 are other than Gln (Q), or the residue at position 6 is otherthan Gln (Q) while the residue at position 22 is absent.

Exemplary peptides that share the consensus structure with SEQ ID NO: 47or 12, or are derived from one of SEQ ID NOS: 47 and 12, and meet theconsensus structure of SEQ ID NO: 93, are identified in Table 3 below:

TABLE 3 Peptide Variants of Peptide P15/P20 Consensus(SEQ ID NOS: 47 or 12) SEQ Peptide ID Name Sequence NO: WildtypeQKDVNFGTPDSTVQNPQDASKPNDSQSNIAKLISALIMSLLQMLT 48 P15b                    KPNDSQSNIAKLISALIMSLLQ 49 P15b-8D-18E                    KPNDSQSDIAKLISALIESLLQ 50 Pl5b-8D-18A                    KPNDSQSDIAKLISALIASLLQ 51 P15b-8D-18S                    KPNDSQSDIAKLISALISSLLQ 52 P15b-8D-18T                    KPNDSQSDIAKLISALITSLLQ 53 P15b-8D-18K                    KPNDSQSDIAKLISALIKSLLQ 54 P15b-8D-6,18E                    KPNDSESDIAKLISALIESLLQ 55 P15b-3,8D                    KPDDSQSDIAKLISALIMSLLQ 56 P15b-3,8D-6E                    KPDDSESDIAKLISALIMSLLQ 57 P15b-3,8D-18E                    KPDDSQSDIAKLISALIESLLQ 58 P15b-3,8D-6,18E                    KPDDSESDIAKLISALIESLLQ 59 P15b-3,8D-allE                    KPDDSESDIAKLISALIESLLE 60 P15b-8D-allE                    KPNDSESDIAKLISALIESLLE 61 P15b-3D-allE                    KPDDSESNIAKLISALIESLLE 62 P15a    NFGTPDSTVQNPQDASKPNDSQSNIAKLISALIMSLLQM 63 P15a-34Q-39P    NFGTPDSTVQNPQDASKPNDSQSNIAKLISALIQSLLQP 142 P15a-39P    NFGTPDSTVQNPQDASKPNDSQSNIAKLISALIMSLLQP 143 P15a-34Q    NFGTPDSTVQNPQDASKPNDSQSNIAKLISALIQSLLQM 144 P15                    KPNDSQSNIAKLISALIMSLLQM 64 P15-59G                    SEEEEEGGIAKLISALIESLLE 149 P15-59                      SEEEEEIAKLISALIESLLE 150 P15-dn4                        SQSNIAKLISALIMSLLQ 227 P20      GTPDSTVQNPQDASKPNDSQSNIAKLIS_LIMSLL 65 P20-5                    KPNDSQSNIAKLIS_LIMSLL 151 P20-6                    KPNDSQSNIAKLIS_LIESLL 152Select peptides in Table 3 include solubility tags, indicated by italicprint, including SEEEEE. Peptides comprising the sequences shown inTable 3 but lacking this specific solubility tag (or having a differentsolubility tag) are also contemplated herein.

In this embodiment, the corresponding wildtype amino acid sequencecorresponds to amino acids 52 to 96 of the Pseudomonas syringae HrpWsequence identified in PCT Application WO 01/98501 to Fan et al., whichis hereby incorporated by reference in its entirety. For purposes ofcomparing properties of the inventive peptides, it is intended that thepolypeptide comprising or the peptide consisting of the amino acidsequence of SEQ ID NO: 48 is used as a reference.

In certain embodiments, the peptide includes one or more mutationsrelative to the corresponding wildtype amino acid sequence of SEQ ID NO:48. These one or more mutations include deletions or substitutionsrelative to SEQ ID NO: 48. In certain embodiments, the one or moremutations improve the solubility in aqueous solution, stability, and/orresistance to chemical degradation of the isolated peptide relative to apolypeptide comprising or consisting of the corresponding wildtype aminoacid sequence of SEQ ID NO: 48. In this embodiment, the isolated peptideis stable when dissolved in water; resistant to chemical degradation inaqueous conditions in the presence of a pH buffer and a biocide, asdescribed above; and/or has a solubility in an aqueous solution of atleast about 1.0 mg/ml.

In certain embodiments, an isolated peptide comprising the amino acidsequence of SEQ ID NO: 47 is a peptide that has an overall lengthbetween 20 and 36 amino acids, and consists essentially of SEQ ID NO:49, SEQ ID NO: 63, or SEQ ID NO: 64, whereas in another embodiment theisolated peptide consists of SEQ ID NO: 49, SEQ ID NO: 63, or SEQ ID NO:64.

In certain embodiments, an isolated peptide comprising the amino acidsequence of SEQ ID NO: 47 is a peptide that has an overall length ofless than 100 amino acids and the amino acid sequence includes SEQ IDNO: 65. In certain embodiments the amino acid sequence of the peptideconsists essentially of SEQ ID NO: 65, whereas in another embodiment theisolated peptide consists of SEQ ID NO: 65.

A further aspect of the invention relates to an isolated peptide havingthe amino acid sequence of:

-   (i) PSPXTXXLXXIVGXILXAXN (SEQ ID NO: 66, P6/6a consensus), wherein    -   X at position 4 is F or Y;    -   X at position 6 is Q, E, g-glutamate, G, A, or S;    -   X at position 7 is optional and, according to one embodiment,        can be M, E, g-glutamate, G, A, S, T, or K; or according to        another embodiment can be L;    -   X at position 9 is M, E, g-glutamate, G, A, S, T, or K;    -   X at position 10 is H or N;    -   X at position 14 is E, g-glutamate, D, or isoD;    -   X at position 17 is Q, E, g-glutamate, G, A, or S; and    -   X at position 19 is Q, E, g-glutamate, G, A, or S; or-   (ii) XTXXLXXIVGXIL (SEQ ID NO: 135, P6/6a min consensus), wherein    -   X at position 1 is F or Y;    -   X at position 3 is Q, E, g-glutamate, G, A, or S;    -   X at position 4 is optional and, according to one embodiment,        can be M, E, g-glutamate, G, A, S, T, or K; or according to        another embodiment can be L;    -   X at position 6 is M, E, g-glutamate, G, A, S, T, or K;    -   X at position 7 is H or N; and    -   X at position 11 is E, g-glutamate, D, or isoD;        wherein the isolated peptide comprises one or more mutations        relative to a corresponding wildtype amino acid sequence. In        certain embodiments, the one or more mutations improve the        aqueous solubility, stability, or resistance to chemical        degradation of the isolated peptide relative to a polypeptide        comprising the corresponding wildtype amino acid sequence.

A comparative wildtype sequence corresponds to amino acids 85-105 of thefull length harpin of Xanthomonas oryzae pv. oryzae (Ji et al., “TwoCoiled-Coil Regions of Xanthomonas oryzae pv. Oryzae Harpin Differ inOligomerization and Hypersensitive Response Induction,” Amino Acids40:381-392 (2011), which is hereby incorporated by reference in itsentirety). This comparative wildtype sequence is the peptide consistingof the amino acid sequence PSPFTQMLMHIVGEILQAQNG (SEQ ID NO: 153).

In certain embodiments, the peptide according to this aspect does notconsist of the amino acid sequence of PSPFTQMLMHIVGEILQAQN (P6a, SEQ IDNO: 67), which corresponds to amino acids 85-104 of the full lengthharpin of Xanthomonas oryzae pv. oryzae (Ji et al., “Two Coiled-CoilRegions of Xanthomonas oryzae pv. Oryzae Harpin Differ inOligomerization and Hypersensitive Response Induction,” Amino Acids40:381-392 (2011), which is hereby incorporated by reference in itsentirety).

In certain embodiments, the peptide of this aspect does not comprise thepeptide sequence of motif 2 as described in U.S. Pat. No. 8,440,881,which is defined as(P/A/V)S(P/Q/A)(F/L/Y)TQ(M/A)LM(H/N/Q)IV(G/M)(E/D/Q), SEQ ID NO: 154. Byway of example, peptides according to SEQ ID NO: 66 do not includepeptides having M/A/T at position 7 when all other aligning residuesmatch the sequence of motif 2; or peptides according to SEQ ID NO: 66 donot include peptides having H/N at position 10 when all other aligningresidues match the sequence of motif 2; or peptides according to SEQ IDNO: 66 do not include peptides having E/D at position 14 when all otheraligning residues match the sequence of motif 2. Similarly, peptidesaccording to SEQ ID NO: 135 do not include peptides having M/A/T atposition 4 when all other aligning residues match the sequence of motif2; or peptides according to SEQ ID NO: 135 do not include peptideshaving H/N at position 7 when all other aligning residues match thesequence of motif 2; or peptides according to SEQ ID NO: 135 do notinclude peptides having E/D at position 11 when all other aligningresidues match the sequence of motif 2.

In certain embodiments, the peptide includes one or more mutationsrelative to the corresponding wildtype amino acid sequence of SEQ ID NO:153. These one or more mutations include deletions or substitutionsrelative to SEQ ID NO: 153. In certain embodiments, the one or moremutations improve the solubility in aqueous solution, stability, and/orresistance to chemical degradation of the isolated peptide relative to apolypeptide comprising or consisting of the corresponding wildtype aminoacid sequence of SEQ ID NO: 153.

The length of peptides according to this aspect is preferably less thanabout 100 amino acids, or alternatively less than 90 amino acids, lessthan 80 amino acids, less than 70 amino acids, less than 60 amino acids,or less than about 50 amino acids. In certain embodiments, the peptideis between 19 and 50 amino acids in length.

In certain embodiments, the peptides according to SEQ ID NOS: 66 and 135also meet the structural features defining the peptides of SEQ ID NO:93, in which case methionine and cysteine residues are not present. Forexample, in peptides according to SEQ ID NO: 66 that are free ofmethionine amino acid residues, X at position 7, if present, is E,g-glutamate, G, A, S, T, K, or L; and X at position 9 is E, g-glutamate,G, A, S, T, or K. Similarly, for peptides according to SEQ ID NO: 135that are free of methionine amino acid residues, X at position 4, ifpresent, is E, g-glutamate, G, A, S, T, K, or L; and X at position 6 isE, g-glutamate, G, A, S, T, or K.

In certain embodiments, the peptides sharing the structure of SEQ ID NO:66 have at least one of the residues at positions 6, 17, and 19 of SEQID NO: 66 being other than Gln (Q), i.e., being E, g-glutamate, G, A, orS. In certain embodiments, two or three of the residues at positions 6,17, and 19 of SEQ ID NO: 66 are other than Gln (Q). Similarly, forpeptides sharing the structure of SEQ ID NO: 135, according to oneembodiment these peptides have the residue at positions 6 being otherthan Gln (Q), i.e., being E, g-glutamate, G, A, or S.

Exemplary peptides that share the consensus structure with SEQ ID NO: 66or135, or are derived from one of SEQ ID NOS: 66 and 135, and meet theconsensus structure of SEQ ID NO: 93, are identified in Table 4 below:

TABLE 4 Peptide Variants of Peptide P6/P6b Consensus(SEQ ID NO: 66 or 135) SEQ Peptide ID Name Sequence NO: wildtype   PSPFTQMLMHIVGEILQAQNG 153 P6a    PSPFTQMLMHIVGEILQAQN 67 P6   PSPFTQ_LMHIVGEILQAQN 68 P6a-7A    PSPFTQALMHIVGEILQAQN 69 P6a-lON   PSPFTQMLMNIVGEILQAQN 70 P6a-4Y    PSPYTQMLMHIVGEILQAQN 71 P6a-14D   PSPFTQMLMHIVGDILQAQN 72 P6a-7,9A    PSPFTQALAHIVGEILQAQN 73 P6a-6E-7A   PSPFTEALMHIVGEILQAQN 74 P6a-6,17E-7A    PSPFTEALMHIVGEILEAQN 75P6a-allE-7A    PSPFTEALMHIVGEILEAEN 76 P6a-allE-4Y-7A   PSPYTEALMHIVGEILEAEN 77 P6a-allE-7A-10N    PSPFTEALMNIVGEILEAEN 78P6a-allE-7A-14D    PSPFTEALMHIVGDILEAEN 79 P6a-allE-4Y-7A-   PSPYTEALMNIVGDILEAEN 80 10N-14D P6b       FTQMLMHIVGEILQAQN 155 P6c   PSPFTQMLMHIVGEIL 156 P6-7L    PSPFTQLLMHIVGEILQAQN 157 P6-9E   PSPFTQMLEHIVGEILQAQN 158 P6a-7L,9E    PSPFTQLLEHIVGEILQAQN 159 P6d SEEEEEFTQMLMHIVGEIL 160 P6d-7L-9E  SEEEEEFTQLLEHIVGEIL 161 p6a-dN3-sol    SEEFTQMLMHIVGEILQAQN 197 p6a-dC4-sol SEEEPSPFTQMLMHIVGEIL 198Select peptides in Table 4 include solubility tags, indicated by italicprint, including SEE, SEEE, and SEEEEE. Peptides comprising thesequences shown in Table 4 but lacking these specific solubility tags(or having a different solubility tag) are also contemplated herein.

Another aspect of the invention relates to a peptide having the aminoacid sequence of:

-   (i) XXXXXXXXXXX(L/M)XXLLXXLLXXLLXXX (SEQ ID NO: 18, P17/18), wherein    -   X at position 1 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 2 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 3 can be any amino acid, but preferably P, Q, S,        E, g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 4 can be any amino acid, but preferably I, Q, S,        E, g-glutamate, A, T, G, D, N, isoD, K, or R;    -   X at position 5 can be any amino acid, but preferably D, isoD,        S, E, g-glutamate, A, T, G, N, Q, K, or R;    -   X at position 6 can be any amino acid, but preferably R, Q, S,        E, g-glutamate, A, T, G, D, isoD, N, or K;    -   X of position 7 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 8 can be any amino acid, but preferably T, Q, S,        E, g-glutamate, A, G, D, isoD, N, K, or R;    -   X at position 9 can be any amino acid, but preferably I, Q, S,        E, g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 10 can be any amino acid, but preferably E,        g-glutamate, Q, S, A, T, G, D, isoD, N, K, or R;    -   X at position 11 can be any amino acid, but preferably Q, S, E,        g-glutamate, A, T, G, D, isoD, N, K, or R;    -   X at position 13 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 14 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 17 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 18 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 21 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 22 can be any amino acid, but preferably S, A,T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 25 can be any amino acid, but preferably S, A, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 26 can be any amino acid, but preferably P, S, A,        T, G, D, isoD, E, g-glutamate, Q, N, K, or R; and    -   X at position 27 can be any amino acid, but preferably Q, S, A,        T, G, D, isoD, E, g-glutamate, N, K, or R; or-   (ii) (L/M)XXLLXXLLXXLL (SEQ ID NO: 25, P17/18 min consensus),    wherein    -   X at position 2 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 3 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 6 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 7 can be any amino acid, but preferably Q, A, S,        T, G, D, isoD, E, g-glutamate, N, K, or R;    -   X at position 10 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R; and    -   X at position 11 can be any amino acid, but preferably S, A,T,        G, D, isoD, E, g-glutamate, Q, N, K, or R.

In certain embodiments, the peptide includes one or more mutationsrelative to a corresponding wildtype amino acid sequence of Erwiniaamylovora HrpW. These one or more mutations include deletions orsubstitutions relative to the wildtype HrpW sequence. In certainembodiments, the one or more mutations improve the solubility in aqueoussolution, stability, and/or resistance to chemical degradation of theisolated peptide relative to a polypeptide comprising or consisting ofthe corresponding wildtype amino acid sequence of Erwinia amylovoraHrpW.

PCT Application WO 01/98501 to Fan et al., which is hereby incorporatedby reference in its entirety, identifies two hypersensitive responseeliciting domains of HrpW_(Ea). The first extends from amino acid 5 toamino acid 64, particularly from amino acid 31 to amino acid 57 ofHrpW_(Ea). The second domain extends from amino acid 103 to amino acid146, particularly from amino acid 116 to amino acid 140 of HrpW_(Ea).Despite this description in Fan et al., the reference identifies only asingle peptide fragment of HrpW_(Ea), which is the peptide consisting ofamino acids 10 to 59.

A comparative wildtype sequence corresponds to amino acids 10 to 59 ofthe full length Erwinia amylovora HrpW sequence identified in PCTApplication WO 01/98501 to Fan et al., which is hereby incorporated byreference in its entirety. For purposes of comparing properties of theinventive peptides, it is intended that the peptide consisting of aminoacids 10 to 59 of the Erwinia amylovora HrpW is used as a reference.

In certain embodiments, the peptide of this aspect does not consist ofthe amino acid sequenceTSSSPGLFQSGGDNGLGGHNANSALGQQPIDRQTIEQMAQLLAELLKSLL (SEQ ID NO: 162),which corresponds to amino acids 10 to 59 of the full length Erwiniaamylovora HrpW (PCT Application WO 01/98501 to Fan et al., which ishereby incorporated by reference in its entirety).

In certain embodiments, the peptide includes one or more mutationsrelative to the corresponding wildtype amino acid sequence of SEQ ID NO:162. These one or more mutations include deletions or substitutionsrelative to SEQ ID NO: 162. In certain embodiments, the one or moremutations improve the solubility in aqueous solution, stability, and/orresistance to chemical degradation of the isolated peptide relative to apolypeptide comprising or consisting of the corresponding wildtype aminoacid sequence of SEQ ID NO: 162.

The length of peptides according to this fourth aspect is preferablyless than about 100 amino acids, or alternatively less than 90 aminoacids, less than 80 amino acids, less than 70 amino acids, less than 60amino acids, or less than about 50 amino acids. In certain embodiments,the peptide is between 13 and 50 amino acids in length, or even between13 and 40 amino acids in length.

In certain embodiments, the peptides according to SEQ ID NOS: 18 and 25also meet the structural features defining the peptides of SEQ ID NO:93, in which case methionine and cysteine residues are not present. Forexample, when the peptide comprising SEQ ID NO: 18 is free of methionineamino acid residues, the amino acid at position 12 is L. Similarly, whenthe peptide comprising SEQ ID NO: 25 is free of methionine amino acidresidues, the amino acid at position 1 is L.

In certain other embodiments, one or more of amino acids 1 to 11 and/or25 to 27 is not present in the isolated peptide of SEQ ID NO: 18. Forexample, peptides lacking amino acids 25 to 27 exhibit improvedstability relative to the wildtype sequence.

Exemplary peptides that share the consensus structure with SEQ ID NO: 18or 25, or are derived from one of SEQ ID NOS: 18 and 25, or meet theconsensus structure of SEQ ID NO: 93, are identified in Table 5 below:

TABLE 5 Peptide Variants of Peptides P17/P18 Consensus(SEQ ID NO: 18 or 25) SEQ Peptide ID Name Sequence NO: wild-[*]QQPIDRQTIEQMAQLLAELLKSLL 162 type P17 [*]QQPIDRQTIEQMAQLLAQLLKSLL 81P17a [*]QQPIDRQTIEQLAQLLAQLLKSLL 82 P18    QQPIDRQTIEQMAQLLAQLLKSLLSPQ83 P18a    QQPIDRQTIEQLAQLLAQLLKSLLSPQ 84 P18b           IEQMAQLLAQLLKSLL 85 P18c            IEQLAQLLAQLLKSLL 86 P18d       DRQTIEQMAQLLAQLLKSLL 87 P18e        DRQTIEQLAQLLAQLLKSLL 88 P18-1   QQPIDRQTIEQMAQLLAQLLKSLL 163 P18-3    QQPIDRQTIEQLAQLLAQLLKSLLSP 228P18-4        DRQTIEQLAQLLAQLLKSLLSP 164 P18-5         QTIEQLAQLLAQLLKSLLSP 165 P18-6      SEEEEEIEQLAQLLAQLLKSLL 166P18-7     SEEEEELAQLLAQLLKSLL 167 P18-10     SEEEEELAELLAELLKSLL 231 InP17, P17a, and the wildtype sequence, [*] = TSSSPGLFQSGGDNGLGGHNANSALGSelect peptides in Table 5 include solubility tags, indicated by italicprint, including SEEEEE. Peptides comprising the sequences shown inTable 5 but lacking these specific solubility tags (or having adifferent solubility tag) are also contemplated herein.

In this embodiment, the wildtype amino acid sequence corresponds toamino acids 10 to 59 of the Erwinia amylovora HrpW sequence identifiedin PCT Application WO 01/98501 to Fan et al., which is herebyincorporated by reference in its entirety. For purposes of comparingproperties of the inventive peptides, it is intended that the peptideconsisting of amino acids 10 to 59 of the Erwinia amylovora HrpW is usedas a reference.

A further aspect of the invention relates to a peptide having the aminoacid sequence of:

XLXX(L/M)LXLIXX(L/I/V/F/M)(L/I/V/F/M) (SEQ ID NO: 26, P19 consensus),wherein

-   -   X at position 1 is optional and can be L, I, V, F, or M;    -   X at position 3 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 4 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R;    -   X at position 7 can be any amino acid, but preferably K, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or R;    -   X at position 10 can be any amino acid, but preferably A, S, T,        G, D, isoD, E, g-glutamate, Q, N, K, or R; and    -   X at position 11 can be any amino acid, but preferably R, A, S,        T, G, D, isoD, E, g-glutamate, Q, N, or K.

As noted above, PCT Application WO 01/98501 to Fan et al., which ishereby incorporated by reference in its entirety, identifies twohypersensitive response eliciting domains of HrpW_(Ea), one of whichextends from amino acid 103 to amino acid 146, particularly from aminoacid 116 to amino acid 140 of HrpW_(Ea). Despite this description in Fanet al., this reference does not identify a peptide fragment of HrpW_(Ea)containing this domain.

A comparative wildtype sequence corresponds to amino acids 116 to 140 ofthe full length Erwinia amylovora HrpW sequence identified in PCTApplication WO 01/98501 to Fan et al., which is hereby incorporated byreference in its entirety. For purposes of comparing properties of theinventive peptides, it is intended that the peptide consisting of aminoacids 116 to 140 of the Erwinia amylovora HrpW is used as a reference.

In certain embodiments, the peptide of this aspect does not consist ofthe amino acid sequence ITPDGQGGGQIGDNPLLKAMLKLIA (SEQ ID NO: 89), whichcorresponds to amino acids 116 to 140 of the full length Erwiniaamylovora HrpW (PCT Application WO 01/98501 to Fan et al., which ishereby incorporated by reference in its entirety).

In certain embodiments, the peptide includes one or more mutationsrelative to the corresponding wildtype amino acid sequence of SEQ ID NO:89. These one or more mutations include deletions or substitutionsrelative to SEQ ID NO: 89. In certain embodiments, the one or moremutations improve the solubility in aqueous solution, stability, and/orresistance to chemical degradation of the isolated peptide relative to apolypeptide comprising or consisting of the corresponding wildtype aminoacid sequence of SEQ ID NO: 89.

The length of peptides according to this aspect is preferably less thanabout 100 amino acids, or alternatively less than 90 amino acids, lessthan 80 amino acids, less than 70 amino acids, less than 60 amino acids,or less than about 50 amino acids. In certain embodiments, the peptideis between 18 and 50 amino acids in length.

Exemplary peptides that share the consensus structure with SEQ ID NO:26, or are derived from SEQ ID NO: 26 and meet the consensus structureof SEQ ID NO: 93, are identified in Table 6 below:

TABLE 6 Peptide Variants of Peptide P19 Consensus (SEQ ID NO: 26) SEQPeptide ID Name Sequence NO: P19 ITPDGQGGGQIGDNPLLKAMLKLIA 89 P19-ITPDGQGGGQIGDNPLLKALLKLIA 90 20L P19a ITPDGQGGGQIGDNPLLKAMLKLIARMMDG 91P19a- ITPDGQGGGQIGDNPLLKALLKLIARLLDG 92 allL P19-4     QGGGQIGDNPLLKAMLKLIARMMDG 226 P19-5     SEEEEEIGDNPLLKALLKLIARLLDG168 P19-5a     SEEEEEIGDDELLKALLKLIARLLDG 169 P19-6         SEEEEELLKALLKLIARLLDG 170 P19-11     SEEEEEIGDNPLLKALLKLIARLL171 P19-7          SEEEEELLKALLKLIARLL 172 P19-8          SEEEEELKALLKLIARLL 173

Select peptides in Table 6 include solubility tags, indicated by italicprint, including SEEEEE. Peptides comprising the sequences shown inTable 6 but lacking this specific solubility tag (or having a differentsolubility tag) are also contemplated herein.

Certain peptides in Table 6 also meet the structural features definingthe peptides of SEQ ID NO: 93, in which case methionine and cysteineresidues are not present. When these peptides also meet the limitationsof SEQ ID NO: 93, amino acid residue 1 of SEQ ID NO: 26, when present,is L, I, V, or F; amino acid 5 of SEQ ID NO: 26 is L; and amino acids 12and 13 of SEQ ID NO: 26 are independently L, I, V, or F.

Still another aspect of the invention relates to a peptide having theamino acid sequence:

-   (i) LXXLLXXLVXLLK (SEQ ID NO: 13, P14d consensus), wherein    -   X at position 1 can be: Q, N, D, E, g-glutamate, isoD, or S;    -   X at position 2 can be: D, E, g-glutamate, isoD;    -   X at position 3 can be: P, D, E, isoD, or g-glutamate;    -   X at position 4 can be M, A, S, D, E, isoD, or g-glutamate    -   X at position 5 can be Q, E, or g-glutamate;    -   X at position 6 can be A, E, or g-glutamate;    -   X at position 8 can be M, L, E, Q, D, N, G, A, S, isoD, or        g-glutamate;    -   X at position 9 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 12 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 13 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate; and    -   X at position 16 can be K, Q, N, E, D, R, G, A, or S; or-   (ii) LXXLLXXLVXLLK (SEQ ID NO: 14, P14d min consensus), wherein    -   X at position 2 can be M, L, E, Q, D, N, G, A, S, isoD, or        g-glutamate;    -   X at position 3 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 6 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate;    -   X at position 7 can be Q, N, E, D, G, A, S, isoD, or        g-glutamate; and    -   X at position 10 can be K, Q, N, E, D, R, G, A, or S.

In certain embodiments, the peptide includes one or more mutationsrelative to the corresponding wildtype amino acid sequence of Ralstoniasolanacearum (previously Pseudomonas solanacearum) PopA. These one ormore mutations include deletions or substitutions relative to thewildtype PopA sequence. In certain embodiments, the one or moremutations improve the solubility in aqueous solution, stability, and/orresistance to chemical degradation of the isolated peptide relative to apolypeptide comprising or consisting of the corresponding wildtype aminoacid sequence of Ralstonia solanacearum PopA.

A comparative wildtype sequence corresponds to amino acids 92 to 125 ofthe Ralstonia solanacearum (previously Pseudomonas solanacearum) PopAsequence identified in PCT Application WO 01/98501 to Fan et al., whichis hereby incorporated by reference in its entirety. For purposes ofcomparing properties of the inventive peptides, it is intended that thewildtype peptide of Fan et al., consisting of amino acids 92 to 125 ofthe Ralstonia solanacearum PopA, is used as a reference.

In certain embodiments, the peptide of this aspect does not consist ofthe amino acid sequence of QAPQSANKTGNVDDANNQDPMQALMQLLEDLVKL (SEQ IDNO: 174), which corresponds to amino acids 92 to 125 of the Ralstoniasolanacearum PopA (see PCT Application WO 01/98501 to Fan et al., whichis hereby incorporated by reference in its entirety).

The length of peptides according to this aspect is preferably less thanabout 100 amino acids, or alternatively less than 90 amino acids, lessthan 80 amino acids, less than 70 amino acids, less than 60 amino acids,or less than about 50 amino acids. In certain embodiments, the peptideis between 12 and 50 amino acids in length.

Exemplary peptides that share the consensus structure with SEQ ID NOS:13 or 14, or are derived from SEQ ID NO: 13 and meet the consensusstructure of SEQ ID NO: 93, are identified in Table 7 below:

TABLE 7 Peptide Variants of Peptide P14d (SEQ ID NO: 13) SEQ Peptide IDName Sequence NO: wildtype  QAPQSANKTGNVDDANNQDPMQALMQLLEDLVKL 174 P14d                  QDPMQALMQLLEDLVKLLK 175 P14e                  QDPAQALLQLLEDLVKLLK 176 P14f                  QDPAQALEQLLEDLVKLLK 177 P14-30                 SEEEEEALEQLLEDLVKLLK 178 P14cQAGPQSANKTGNVDDANNQDPMQALMQLLEDLVKLLK 199Select peptides in Table 7 include solubility tags, indicated by italicprint, including SEEEEE. Peptides comprising the sequences shown inTable 7 but lacking this specific solubility tag (or having a differentsolubility tag) are also contemplated herein.

It is notable that a C-terminal lysine residue seems to be necessary forHR elicitation by p14d variants. This is a slight deviation from thecanonical sequence of SEQ ID NO: 93. Without being bound by belief, itis believed that the C-terminal lysine may be necessary due to thesingle hydrophilic amino acid between 2 hydrophobic doublet sequenceswithin the p14d variants (LVKLL).

Certain peptides according to this aspect also meet the structuralfeatures defining the peptides of SEQ ID NO: 93, in which casemethionine and cysteine residues are not present. For example, forpeptides comprising SEQ ID NO: 13, amino acid residue 4 of SEQ ID NO: 13is A, S, D, isoD, E, or g-glutamate, and amino acid residue 8 of SEQ IDNO: 13 is L, E, g-glutamate, Q, D, isoD, N, G, A, or S. Similarly, forpeptides comprising SEQ ID NO: 14 the amino acid residue at position 2is L, E, g-glutamate, Q, D, isoD, N, G, A, or S.

Yet another aspect of the invention relates to a peptide having theamino acid sequence:

-   (i) LXXL(L/M)XILXXLV (SEQ ID NO: 16, P25 consensus) wherein    -   X at position 2 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G;    -   X at position 3 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 6 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 9 can be E, g-glutamate, D, isoD, Q, N, T, S, A,        or G; and    -   X at position 10 can be A, G, S, T, E, g-glutamate, D, isoD, Q,        or N; or-   (ii) LXXVLXXL(L/M)XILXXLV (SEQ ID NO: 17, P25 consensus) wherein    -   X at position 2 can be T, S, A, G, D, isoD, E, g-glutamate, Q,        or N;    -   X at position 3 can be G, T, S, A, D, isoD, E, g-glutamate, Q,        or N;    -   X at position 6 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G;    -   X at position 7 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 10 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 13 can be E, g-glutamate, D, isoD, Q, N, T, S, A,        or G;    -   X at position 14 can be A, G, S, T, E, g-glutamate, D, isoD, Q,        or N; and    -   V at position 16 is optional.

In certain embodiments, the peptide includes one or more mutationsrelative to a corresponding wildtype amino acid sequence of Ralstoniasolanacearum (previously Pseudomonas solanacearum) PopA. These one ormore mutations include deletions or substitutions relative to thewildtype PopA sequence. In certain embodiments, the one or moremutations improve the solubility in aqueous solution, stability, and/orresistance to chemical degradation of the isolated peptide relative to apolypeptide comprising or consisting of the corresponding wildtype aminoacid sequence of Ralstonia solanacearum PopA.

A comparative wildtype sequence corresponds to amino acids 206 to 260 ofthe Ralstonia solanacearum (previously Pseudomonas solanacearum) PopAsequence, which is identified in PCT Application WO 01/98501 to Fan etal., which is hereby incorporated by reference in its entirety, as ahypersensitive response domain. For purposes of comparing properties ofthe inventive peptides, it is intended that the wildtype peptide of Fanet al., consisting of amino acids 206 to 260 of the Ralstoniasolanacearum PopA, is used as a reference.

In certain embodiments, the peptide of this aspect does not consist ofthe amino acid sequence of NGADGGNGVNGNQANGPQNAGDVNGANGADDGSEDQGGLTGVLQKLMKILNALVQ (SEQ ID NO: 179), which corresponds to amino acids 206 to 260of the Ralstonia solanacearum PopA (see PCT Application WO 01/98501 toFan et al., which is hereby incorporated by reference in its entirety).

The length of peptides according to this aspect is preferably less thanabout 100 amino acids, or alternatively less than 90 amino acids, lessthan 80 amino acids, less than 70 amino acids, less than 60 amino acids,or less than about 50 amino acids. In certain embodiments, the peptideis between 12 and 50 amino acids in length.

Exemplary peptides that share the consensus structure with one of SEQ IDNOS: 16 or 17, or are derived from one of SEQ ID NOS: 16 or 17 and meetthe consensus structure of SEQ ID NO: 93, are identified in Table 8below:

TABLE 8 Peptide Variants of Peptides P2(SEQ ID NO: 180) and P25 (SEQ ID NO: 182) SEQ Peptide ID Name SequenceNO: wildtype [*]ANGADDGSEDQGG__LTGVLQKLMKILNALVQ 179 P2   ANGADDGSEDQGGLTLTGVLQKLMKILNALVQ 180 P25-4           EDQGGLTLTGVLQKLMKILNALVQ 181 P25              GGLTLTGVLQKLMKILNAL 182 P25s           EDQGGLTLTGVLQKLMKILNAL 183 P25-7           EDQGGLTLTGVLQKLLKILNAL 184 P25-8           EDQGGLILTGVLQELMEILNAL 185 P25-20E           EDQGGLTLTGVLQKLLKILEALVQ 186 P25-10           SEEEELTLTGVLQKLLKILEAL 187 P25-11            SEEEEELTGVLQKLLKILEAL 188 P25-15          SEEEEELTLTGVLQKLLKILEA 200 P25-16               SEEEEEVLQKLLKILEALV 201 P25-17                SEEEEELQKLLKILEALVQ 202 [*] = N-terminal sequenceNGADGGNGVNGNQANGPQNAGDVNGSelect peptides in Table 8 include solubility tags, indicated by italicprint, including SEEEEE. Peptides comprising the sequences shown inTable 8 but lacking these specific solubility tags (or having adifferent solubility tag) are also contemplated herein.

Notably, a number of these derivative peptides in Table 8 include arepeated LT sequence not observed in the wildtype sequence. However, oneshould note that these sequences require a larger hydrophobic sequenceto cause a hypersensitive response as compared with SEQ ID NO: 93.Without being bound by belief, it is believed that this may be due tothe presence of the amino acid valine in the sequence rather thanleucine as well as the presence of only a single hydrophilic amino acidbetween the hydrophobic doublets (LLKIL). Although these changes aredeleterious to HR, their effect can be reversed by the addition ofadditional hydrophobic residues at the C-terminus of the peptide ( . . .KIL versus . . . KILEALV or . . . KILNALV).

Certain peptides according to this aspect also meet the structuralfeatures defining the peptides of SEQ ID NO: 93, in which casemethionine and cysteine residues are not present. For example, forpeptides comprising SEQ ID NO: 16, amino acid residue 5 is L; and forpeptides comprising SEQ ID NO: 17, amino acid residue 9 is L.

Yet another aspect of the invention relates to a peptide having theamino acid sequence:

-   (i) (L/M)XXLLX(L/M)FXXI(L/M)XX (SEQ ID NO: 15, P3min consensus)    wherein    -   X at position 2 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G;    -   X at position 3 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G;    -   X at position 6 can be K, Q, N, E, g-glutamate, D, isoD, T, S,        A, or G;    -   X at position 9 can be E, g-glutamate, D, isoD, Q, N, T, S, A,        or G;    -   X at position 10 can be A, G, S, T, E, g-glutamate, D, isoD, Q,        or N;    -   X at position 13 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G; and    -   X at position 14 can be Q, N, E, g-glutamate, D, isoD, T, S, A,        or G.

In certain embodiments, the peptide includes one or more mutationsrelative to a corresponding wildtype amino acid sequence of Erwiniaamylovora HrpN. These one or more mutations include deletions orsubstitutions relative to the wildtype HrpN sequence. In certainembodiments, the one or more mutations improve the solubility in aqueoussolution, stability, and/or resistance to chemical degradation of theisolated peptide relative to a polypeptide comprising or consisting ofthe corresponding wildtype amino acid sequence of Erwinia amylovoraHrpN.

A comparative wildtype sequence corresponds to amino acids 137 to 180 or150 to 180 of the Erwinia amylovora HrpN sequence, which are identifiedin U.S. Pat. No. 7,132,525 to Wei et al., which is hereby incorporatedby reference in its entirety. The HrpN peptide containing aa 137 to 180was identified as a hypersensitive response-eliciting fragment, whereasthe HrpN peptide containing aa 150 to 180 could not be expressed andtested. For purposes of comparing properties of the inventive peptides,it is intended that the wildtype peptide of Wei et al., consisting ofeither amino acids 137 to 180 or 150 to 180 of the Erwinia amylovoraHrpN, is used as a reference.

In certain embodiments, the peptide of this aspect does not consist ofthe amino acid sequence ofS₁₃₇TSQNDDSTSGTDS₁₅₀TSDSSDPMQQLLKMFSEIMQSLFGDGQDGT₁₈₀ (SEQ ID NO: 230),which corresponds to amino acids 137 to 180 of the Erwinia amylovoraHrpN (see U.S. Pat. No. 7,132,525 to Wei et al., which is herebyincorporated by reference in its entirety), or the 31-amino acid peptidecorresponding to aa 150 to 180 thereof.

The length of peptides according to this aspect is preferably less thanabout 100 amino acids, or alternatively less than 90 amino acids, lessthan 80 amino acids, less than 70 amino acids, less than 60 amino acids,less than about 50 amino acids, less than about 40 amino acids, or lessthan 30 amino acids. In certain embodiments, the peptide is between 12and 30 amino acids in length.

Exemplary peptides that share the consensus structure with SEQ ID NOS:15, or are derived from SEQ ID NOS: 15 and meet the consensus structureof SEQ ID NO: 93, are identified in Table 9 below:

TABLE 9 Peptide Variants of Peptide P3 Consensus (SEQ ID NO: 15) PeptideSEQ Name Sequence ID NO: wildtypeSTSQNDDSTSGTDSTSDSSDPMQQLLKMFSEIMQSLFGDGQDGT 203 P3   QNDDSTSGTDSTSDSSDPMQQLLKMFSEIMQSLFGDGQDGT 204 P3-3                  SDPMQQLLKMFSEIMQSLF 205 P3-4                 SEEELQQLLKLFSEILQSLF 206 P3-6               SEEEEELQQLLKLFSEILQSL 207 P3-7               SEEEEELQQLLKLFSEILQS 208 P3-11                     LQQLLKLFSEILQSLFEEEE 209Select peptides in Table 9 include solubility tags, indicated by italicprint, including SEEE, SEEEEE, and EEEE. Peptides comprising thesequences shown in Table 9 but lacking these specific solubility tags(or having a different solubility tag) are also contemplated herein.

It is notable that the minimal P3 sequence requires a longer sequencethan the minimal HR-box sequence of SEQ ID NO: 93. Without being boundby belief, it is believed that this may be due to the presence of twophenylalanine residues within the hydrophobic sequence.

Certain peptides according to this aspect also meet the structuralfeatures defining the peptides of SEQ ID NO: 93, in which casemethionine and cysteine residues are not present. For example, forpeptides comprising SEQ ID NO: 15, amino acid residues 1, 7, and 12 areL.

Based on the disclosed consensus sequence (SEQ ID NO: 93), it ispossible to generate novel peptide sequences with predicted HR activitythat deviate significantly from bacterial protein sequences. Thesepeptides can contain hydrophilic residues optimized for maximumsolubility and chemical stability. In a preferred embodiment, thesehydrophilic residues are glutamate. Lysine and arginine are alsopossible choices, however a large number of these residues will cause atoxic response in the plant.

In addition to the foregoing peptides that are modeled (and modified)based on naturally occurring sequences within larger HR-elicitingproteins, the present invention also contemplates entirely syntheticpeptides that meet the consensus of SEQ ID NO: 93. Ideally, thesesynthetic peptides include a number of strongly hydrophilic amino acidsspanning between the hydrophobic residues specified by SEQ ID NO: 93.Exemplary synthetic peptides are listed in Table 10 below. Thesepeptides contain the necessary hydrophobic peptides associated with HRelicitation. The intervening hydrophilic residues are chosen for maximumsolubility, preferably with charged amino acids. It is possible to useuncharged amino acids, but larger proportions of uncharged amino acidsmay cause the resulting peptide to aggregate in solution and form aprecipitate or gel. Glutamate is preferred for chemical stability overaspartate. Although lysine and arginine have even superior solubilitycharacteristics, poly-cations produced a toxic response in the testedplants. As a result, arginine-rich sequences such as P30-1 (SEQ ID NO:211) should be avoided.

TABLE 10 Other HR-box peptides Peptide SEQ Name Sequence ID NO: P30-2SEELEELLEELIEELL 189 P30-3    LEELLEELIEELLEE 190 P30-4    LEELLEELIEELL210 P30-1   RLRRLLRRLIRRLLRP 211 P30-5    LDDLLDDLIDDLLDD 212 P18-13   LEELLEELLEELLEE 213 P14-54 LEQLLEDLVKLL

EE 214 P14-55 LEQLLEDLVELL

EE 215 P14-56 LEELLEDLVELL

EE 216 P14-57 LEELLEELVELL

EE 217 P3-12 LEELLELFEEILEELFEE 218 P3-13 LEELLKLFEEILEELFEE 219 P20-50aIEELIELIEELLEE 220 P15-67 IEELIEELIEELLEE 221 P19-54c LEELLKLIERLLEE 222P19-54b LEELLELIERLLEE 223 P19-54a LEELLKLIEELLEE 224 P19-54LEELLELIEELLEE 225Select peptides in Table 10 include solubility tags, indicated by italicprint, including SEE, EE, DD, or EEE. Peptides comprising the sequencesshown in Table 10 but lacking these specific solubility tags (or havingdifferent solubility tags) are also contemplated herein.

The isolated peptides of the invention can also be presented in the formof a fusion peptide that includes, in addition, a second amino acidsequence coupled to the inventive peptides via peptide bond. The secondamino acid sequence can be a purification tag, such as poly-histidine(His₆-), a glutathione-S-transferase (GST-), or maltose-binding protein(MBP-), which assists in the purification but can later be removed,i.e., cleaved from the peptide following recovery. Protease-specificcleavage sites or chemical-specific cleavage sites (i.e., in a cleavablelinker sequence) can be introduced between the purification tag and thedesired peptide. Protease-specific cleavage sites are well known in theliterature and include, without limitation, the enterokinase specificcleavage site (Asp)₄-Lys, which is cleaved after lysine; the factor Xaspecific cleavage site Ile-(Glu or Asp)-Gly-Arg, which is cleaved afterarginine; the trypsin specific cleavage site, which cleaves after Lysand Arg; and the Genenase™ I specific cleavage sitePro-Gly-Ala-Ala-His-Tyr. Chemicals and their specific cleavage sitesinclude, without limitation, cyanogen bromide (CNBr), which cleaves atmethionine (Met) residues; BNPS-skatole, which cleaves at tryptophan(Trp) residues; formic acid, which cleaves at aspartic acid-proline(Asp-Pro) peptide bonds; hydroxylamine, which cleaves atasparagine-glycine (Asn-Gly) peptide bonds; and2-nitro-5-thiocyanobenzoic acid (NTCB), which cleaves at cysteine (Cys)residues (see Crimmins et al., “Chemical Cleavage of Proteins inSolution,” Curr. Protocol. Protein Sci., Chapter 11:Unit 11.4 (2005),which is hereby incorporated by reference in its entirety). In order touse one of these cleavage methods, it may be necessary to removeunwanted cleavage sites from within the desired peptide sequences bymutation. For example, p4-7E-cR (SEQ ID NO: 40) has been mutated forcompatibility with trypsin: the lysine residue at position 7 is mutatedto a glutamate and a C-terminal arginine is added to represent theproduct of a theoretical trypsin cleavage. Likewise, p19-5 (SEQ ID NO:168) contains the sequence ‘NP’ which can be cleaved under acidicconditions. Mutation of these residues to ‘DE’ in p19-5a (SEQ ID NO:169) prevents this particular cleavage mechanism. The desired peptideproduct can be purified further to remove the cleaved purification tags.

The isolated peptides of the invention can also be presented in the formof a fusion peptide that includes multiple peptide sequences of thepresent invention linked together by a linker sequence, which may or maynot take the form of a cleavable amino acid sequence of the typedescribed above. Such multimeric fusion proteins may or may not includepurification tags. In one embodiment, each monomeric sequence caninclude a purification tag linked to a peptide of the invention by afirst cleavable peptide sequence; and the several monomeric sequencescan be linked to adjacent monomeric sequences by a second cleavablepeptide sequence. Consequently, upon expression of the multimeric fusionprotein, i.e., in a host cell, the recovered fusion protein can betreated with a protease or chemical that is effective to cleave thesecond cleavable peptide sequence, thereby releasing individualmonomeric peptide sequences containing purification tags. Upon affinitypurification, the recovered monomeric peptide sequences can be treatedwith a protease or chemical that is effective to cleave the firstcleavable peptide sequence and thereby release the purification tag fromthe peptide of interest. The latter can be further purified using gelfiltration and/or HPLC as described infra.

According to one approach, the peptides of the present invention can besynthesized by standard peptide synthesis operations. These include bothFMOC (9-fluorenylmethyloxy-carbonyl) and tBoc (tert-butyloxy-carbonyl)synthesis protocols that can be carried out on automated solid phasepeptide synthesis instruments including, without limitation, the AppliedBiosystems 431 A, 433 A synthesizers and Peptide Technologies Symphonyor large scale Sonata or CEM Liberty automated solid phase peptidesynthesizers. The use of alternative peptide synthesis instruments isalso contemplated. Peptides prepared using solid phase synthesis arerecovered in a substantially pure form.

The peptides of the present invention may be also prepared by usingrecombinant expression systems followed by separation and purificationof the recombinantly prepared peptides. Generally, this involvesinserting an encoding nucleic acid molecule into an expression system towhich the molecule is heterologous (i.e., not normally present). One ormore desired nucleic acid molecules encoding a peptide of the inventionmay be inserted into the vector. The heterologous nucleic acid moleculeis inserted into the expression system or vector in proper sense (5′-3′)orientation and correct reading frame relative to the promoter and anyother 5′ and 3′ regulatory molecules.

Representative nucleotide sequences for expression in bacteria and planthosts are included in Table 11 below:

TABLE 11 Peptide & Optimized SEQ Host Nucleotide Sequence ID NO: P4-14sTCTCAAGGAATTTCTGAAA 145 A. thaliana AGCAACTTGATCAACTTCTTTCTCAACTTATTCAAGCT CTTCTTCAACCT P4-14s AGCCAGGGTATTAGCGAAA 146 E. coliAACAGCTGGATCAGCTGCT GAGCCAGCTGATTCAGGCA CTGCTGCAGCCG P1-2E,8E,AATGAAGGAATTTCTGAAA 147 11E,15E,18E AGGAACTTGATGAACTTCT A. thalianaTACTGAACTTATTGAAGCT CTTCTTCAACAA P1-2E,8E,11E, AATGAAGGTATTAGCGAAA 14815E,18E AAGAACTGGATGAACTGCT E. coli GACCGAACTGATTGAAGCA CTGCTGCAGCAGWith knowledge of the encoded amino acid sequence listed herein and thedesired transgenic organism, additional codon-optimized DNA sequencesand RNA sequences can be generated with nothing more than routine skill.

Expression (including transcription and translation) of a peptide orfusion polypeptide of the invention by the DNA construct may beregulated with respect to the level of expression, the tissue type(s)where expression takes place and/or developmental stage of expression. Anumber of heterologous regulatory sequences (e.g., promoters andenhancers) are available for controlling the expression of the DNAconstruct. These include constitutive, inducible and regulatablepromoters, as well as promoters and enhancers that control expression ina tissue- or temporal-specific manner. Exemplary constitutive promotersinclude the raspberry E4 promoter (U.S. Pat. Nos. 5,783,393 and5,783,394, each of which is hereby incorporated by reference in itsentirety), the nopaline synthase (NOS) promoter (Ebert et al., Proc.Natl. Acad. Sci. (U.S.A.) 84:5745-5749 (1987), which is herebyincorporated by reference in its entirety), the octopine synthase (OCS)promoter (which is carried on tumor-inducing plasmids of Agrobacteriumtumefaciens), the caulimovirus promoters such as the cauliflower mosaicvirus (CaMV) 19S promoter (Lawton et al., Plant Mol. Biol. 9:315-324(1987), which is hereby incorporated by reference in its entirety) andthe CaMV 35S promoter (Odell et al., Nature 313:810-812 (1985), which ishereby incorporated by reference in its entirety), the figwort mosaicvirus 35S-promoter (U.S. Pat. No. 5,378,619, which is herebyincorporated by reference in its entirety), the light-inducible promoterfrom the small subunit of ribulose-1,5-bis-phosphate carboxylase(ssRUBISCO), the Adh promoter (Walker et al., Proc. Natl. Acad. Sci.(U.S.A.) 84:6624-6628 (1987), which is hereby incorporated by referencein its entirety), the sucrose synthase promoter (Yang et al., Proc.Natl. Acad. Sci. (U.S.A.) 87:4144-4148 (1990), which is herebyincorporated by reference in its entirety), the R gene complex promoter(Chandler et al., Plant Cell 1:1175-1183 (1989), which is herebyincorporated by reference in its entirety), the chlorophyll a/b bindingprotein gene promoter, the CsVMV promoter (Verdaguer et al., Plant MolBiol., 37:1055-1067 (1998), which is hereby incorporated by reference inits entirety), and the melon actin promoter (PCT Publ. No. WO00/56863,which is hereby incorporated by reference in its entirety). Exemplarytissue-specific promoters include the tomato E4 and E8 promoters (U.S.Pat. No. 5,859,330, which is hereby incorporated by reference in itsentirety) and the tomato 2AII gene promoter (Van Haaren et al., PlantMol Bio., 21:625-640 (1993), which is hereby incorporated by referencein its entirety).

In one preferred embodiment, expression of the DNA construct is undercontrol of regulatory sequences from genes whose expression isassociated with early seed and/or embryo development. Indeed, in apreferred embodiment, the promoter used is a seed-enhanced promoter.Examples of such promoters include the 5′ regulatory regions from suchgenes as napin (Kridl et al., Seed Sci. Res. 1:209:219 (1991), which ishereby incorporated by reference in its entirety), globulin (Belangerand Kriz, Genet. 129: 863-872 (1991), GenBank Accession No. L22295, eachof which is hereby incorporated by reference in its entirety), gammazein Z 27 (Lopes et al., Mol Gen Genet. 247:603-613 (1995), which ishereby incorporated by reference in its entirety), L3 oleosin promoter(U.S. Pat. No. 6,433,252, which is hereby incorporated by reference inits entirety), phaseolin (Bustos et al., Plant Cell 1(9):839-853 (1989),which is hereby incorporated by reference in its entirety), arcelin5(U.S. Application Publ. No. 2003/0046727, which is hereby incorporatedby reference in its entirety), a soybean 7S promoter, a 7Sa promoter(U.S. Application Publ. No. 2003/0093828, which is hereby incorporatedby reference in its entirety), the soybean 7Sαβ conglycinin promoter, a7Sα promoter (Beachy et al., EMBO J. 4:3047 (1985); Schuler et al.,Nucleic Acid Res. 10(24):8225-8244 (1982), each of which is herebyincorporated by reference in its entirety), soybean trypsin inhibitor(Riggs et al., Plant Cell 1(6):609-621 (1989), which is herebyincorporated by reference in its entirety), ACP (Baerson et al., PlantMol. Biol., 22(2):255-267 (1993), which is hereby incorporated byreference in its entirety), stearoyl-ACP desaturase (Slocombe et al.,Plant Physiol. 104(4):167-176 (1994), which is hereby incorporated byreference in its entirety), soybean a′ subunit of β-conglycinin (Chen etal., Proc. Natl. Acad. Sci. 83:8560-8564 (1986), which is herebyincorporated by reference in its entirety), Vicia faba USP (U.S.Application Publ. No. 2003/229918, which is hereby incorporated byreference in its entirety) and Zea mays L3 oleosin promoter (Hong etal., Plant Mol. Biol., 34(3):549-555 (1997), which is herebyincorporated by reference in its entirety).

Nucleic acid molecules encoding the peptides of the present inventioncan be prepared via solid-phase synthesis using, e.g., thephosphoramidite method and phosphoramidite building blocks derived fromprotected 2′-deoxynucleosides. To obtain the desired oligonucleotide,the building blocks are sequentially coupled to the growingoligonucleotide chain in the order required by the sequence of theproduct. Upon the completion of the chain assembly, the product isreleased from the solid phase to solution, deprotected, collected, andtypically purified using HPLC. The limits of solid phase synthesis aresuitable for preparing oligonucleotides up to about 200 nt in length,which encodes peptides on the order of about 65 amino acids or less. Theends of the synthetized oligonucleotide can be designed to includespecific restriction enzyme cleavage site to facilitate ligation of thesynthesized oligonucleotide into an expression vector.

For longer peptides, oligonucleotides can be prepared via solid phasesynthesis and then the synthetic oligonucleotide sequences ligatedtogether using various techniques. Recombinant techniques for thefabrication of whole synthetic genes are reviewed, for example, inHughes et al., “Chapter Twelve—Gene Synthesis: Methods andApplications,” Methods in Enzymology 498:277-309 (2011), which is herebyincorporated by reference in its entirety.

Once a suitable expression vector is selected, the desired nucleic acidsequences are cloned into the vector using standard cloning proceduresin the art, as described by Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Springs Laboratory, Cold Springs Harbor, N.Y.(1989), or U.S. Pat. No. 4,237,224 to Cohen and Boyer, which are herebyincorporated by reference in their entirety. The vector is thenintroduced to a suitable host.

A variety of host-vector systems may be utilized to recombinantlyexpress the peptides of the present invention. Primarily, the vectorsystem must be compatible with the host used. Host-vector systemsinclude, without limitation, the following: bacteria transformed withbacteriophage DNA, plasmid DNA, or cosmid DNA; microorganisms such asyeast containing yeast vectors; mammalian cell systems infected withvirus (e.g., vaccinia virus, adenovirus, etc.); insect cell systemsinfected with virus (e.g., baculovirus); and plant cells infected byAgrobacterium. The expression elements of these vectors vary in theirstrength and specificities. Depending upon the host-vector systemutilized, any one of a number of suitable transcription and translationelements can be used to carry out this and other aspects of the presentinvention.

Purified peptides may be obtained by several methods. The peptide ispreferably produced in purified form (preferably at least about 80% or85% pure, more preferably at least about 90% or 95% pure) byconventional techniques. Depending on whether the recombinant host cellis made to secrete the peptide into growth medium (see U.S. Pat. No.6,596,509 to Bauer et al., which is hereby incorporated by reference inits entirety), the peptide can be isolated and purified bycentrifugation (to separate cellular components from supernatantcontaining the secreted peptide) followed by sequential ammonium sulfateprecipitation of the supernatant. The fraction containing the peptide issubjected to gel filtration in an appropriately sized dextran orpolyacrylamide column to separate the peptides from other proteins. Ifnecessary, the peptide fraction may be further purified by HPLC.

Alternatively, if the peptide of interest of interest is not secreted,it can be isolated from the recombinant cells using standard isolationand purification schemes. This includes disrupting the cells (e.g., bysonication, freezing, French press, etc.) and then recovering thepeptide from the cellular debris. Purification can be achieved using thecentrifugation, precipitation, and purification procedures describedabove. The use of purification tags, described above, can simplify thisprocess.

In certain embodiments, purification is not required. Where purificationis not performed, cell-free lysates can be recovered followingcentrifugation for removal of cellular debris. The resulting cell-freelysate can be treated with heat for a sufficient amount of time todeactivate any native proteases in the recovered fraction, e.g., 10 minat 100° C. If desired, one or more of biocidal agents, proteaseinhibitors, and non-ionic surfactants can be introduced to such acell-free preparation (see U.S. Application Publ. No. 20100043095 toWei, which is hereby incorporated by reference in its entirety).

Once the peptides of the present invention are recovered, they can beused to prepare a composition that includes a carrier, and one or moreadditives selected from the group consisting of a bacteriocidal orbiocidal agent, a protease inhibitor, a non-ionic surfactant, afertilizer, an herbicide, an insecticide, a fungicide, a nematicide,biological inoculants, plant regulators, and mixtures thereof.

In certain embodiments, the compositions include greater than about 1 nMof the peptide, greater than about 10 nM of the peptide, greater thanabout 20 nM of the peptide, greater than about 30 nM of the peptide,greater than about 40 nM of the peptide, greater than about 50 nM of thepeptide, greater than about 60 nM of the peptide, greater than about 70nM of the peptide, greater than 80 about nM of the peptide, greater thanabout 90 nM of the peptide, greater than about 100 nM of the peptide,greater than about 150 nM of the peptide, greater than about 200 nM ofthe peptide, or greater than about 250 nM of the peptide. In certainembodiments, the compositions include less than about 1 nM of thepeptide. For example, certain peptides can be present at a concentrationof less than about 2 ng/ml, less than about 1.75 ng/ml, less than about1.5 ng/ml, less than about 1.25 ng/ml, less than about 1.0 ng/ml, lessthan about 0.75 ng/ml, less than about 0.5 ng/ml, less than about 0.25ng/ml, or even less than about 0.1 ng/ml.

Suitable carriers include water, aqueous solutions optionally containingone or more co-solvents, slurries, and solid carrier particles.Exemplary solid carriers include mineral earths such as silicates,silica gels, talc, kaolins, limestone, lime, chalk, bole, loess, clays,dolomite, diatomaceous earth, calcium sulfate, magnesium sulfate,magnesium oxide, ground synthetic materials, and products of vegetableorigin, such as cereal meal, tree bark meal, wood meal and nutshellmeal, cellulose powders, starches and starch derivatives, as well asother mono-, di-, and poly-saccharides.

Suitable fertilizers include, without limitation, ammonium sulfate,ammonium phosphate, ammonium nitrate, ureas, and combinations thereof.

Suitable insecticides include, without limitation, members of theneonicotinoid class such as imidicloprid, clothianidin, andthiamethoxam; members of the organophosphate class such as chlorpyrifosand malathion; members of the pyrethroid class such as permethrin; othernatural insecticides such as nicotine, nornicotine, and pyrethrins;members of the carbamate class such as aldicarb, carbofuran, andcarbaryl; members of the macrocyclic lactone class such as variousabamectin, avermectin, and ivermectin products; members of the diamideclass such as chlorantraniliprole, cyantraniliprole, and flubendiamide;chitin synthesis inhibitors, particularly those of the benzoylurea classsuch as lufenuron and diflubenzuron; and any combination thereof,including combinations of two or more, three or more, or four or moreinsecticides. Additional insecticides are listed in the Compendium ofPesticide Common Names, which is database operated by Alan Wood andavailable in electronic form at the alanwood.net internet site.

Suitable fungicides include, without limitation, members of thestrobilurin class such as azoxystrobin, pyraclostrobin, trifloxystrobin,picoxystrobin, and fluoxastrobin; members of the triazole class such asipconazole, metconazole, tebuconazole, triticonazole, tetraconazole,difenoconazole, flutriafol, propiconazole and prothioconazole; membersof the succinate dehydrogenase class such as carboxin, fluxapyroxad,boscalid and sedaxane: members of the phenylamide class such asmetalaxyl, mefenoxam, benalaxyl, and oxadiyxl; members of thephenylpyrrole class such as fludioxonil; members of the phthalimideclass such as captan; members of the dithiocarbamate class such asmancozeb and thiram; members of the benzimidazole class such asthiabendazole; and any combination thereof, including combinations oftwo or more, three or more, or four or more fungicides. Additionalfungicides are listed in the Compendium of Pesticide Common Names, whichis a database operated by Alan Wood and available in electronic form atthe alanwood.net internet site.

Suitable nematicides include, without limitation, chemicals of thecarbamate class such as aldicarb, aldoxycarb, oxamyl, carbofuran, andcleothocarb; and chemicals of the organophosphate class such asthionazin, ethoprophos, fenamiphos, fensulfothion, terbufos, isazofos,and ebufos. Additional nematicides are listed in the Compendium ofPesticide Common Names, which is a database operated by Alan Wood andavailable in electronic form at the alanwood.net internet site.

Suitable bactericides include, without limitation, those based ondichlorophene and benzylalcohol hemi formal (Proxel® from ICI orActicide® RS from Thor Chemie and Kathon® MK from Rohm & Haas) andisothiazolinone derivatives such as alkylisothiazolinones andbenzisothiazolinones (Acticide® MBS from Thor Chemie; Proxel® GXL fromICI). Additional bactericides are listed in the Compendium of PesticideCommon Names, which is a database operated by Alan Wood and available inelectronic form at the alanwood.net internet site.

Suitable inoculants include, without limitation, Bradyrhizobium spp.,particularly Bradyrhizobium japonicum (BASF Vault® products), Bacillussubtilis, Bacillus firmus, Bacillus pumilis, Streptomyces lydicus,Trichoderma spp., Pasteuria spp., other cultures of rhizobial cells(BASF Nodulator® and Rhizo-Flo®), and any combination thereof, includingcombinations of two or more, three or more, or four or more inoculants.

Plant regulators are chemical substances, either natural or synthetic,that either stimulate or inhibit plant biochemical signaling. These areusually, but not exclusively, recognized by receptors on the surface ofthe cell, causing a cascade of reactions in the cell. Suitable plantregulators include, without limitation, ethephon; ethylene; salicylicacid; acetylsalicylic acid; jasmonic acid; methyl jasmonate; methyldihydrojasmonate; chitin; chitosan; abscisic acid; any auxin compound orinhibitor, including but not limited to (4-chlorophenoxy)acetic acid,(2,4-dichlorophenoxy)acetic acid, and 2,3,5-triiodobenzoic acid; anycytokinin, including but not limited to kinetin and zeatin;gibberellins; brassinolide; and any combination thereof, includingcombinations of two or more, three or more, or four or more regulators.

Other suitable additives include buffering agents, wetting agents,coating agents, and abrading agents. These materials can be used tofacilitate application of the compositions in accordance with thepresent invention. In addition, the compositions can be applied to plantseeds with other conventional seed formulation and treatment materials,including clays and polysaccharides.

Compositions or systems use for plant seed treatment include: one ormore of the peptides of the present invention, preferably though notexclusively one of P1, P4-14S, P6a, P14d, P15a, P18, P19, or P25, incombination with one or more insecticides, nematicides, fungicides,other inoculants, or other plant regulators, including combinations ofmultiple insecticides, or multiple nematicides, multiple fungicides,multiple other inoculants, or multiple plant regulators. Suitableinsecticides, nematicides, fungicides, inoculants, and plant regulatorsfor these combination treatments include those identified above. Thesecompositions are presented in the form of a single composition at thetime of seed treatment. In contrast, a system used for seed treatmentmay involve multiple treatments, e.g., a composition containing thepeptides is used in one treatment and a composition containing the oneor more insecticides, nematicides, fungicides, plant regulators and/orbactericides, is used in a separate treatment. In the latter embodiment,both of these treatments are carried out at about the same time, i.e.,before planting or at about the time of planting.

One such example includes one or more of peptides of the presentinvention, including (without limitation) one of P1, P4-14S, P6a, P14d,P15a, P18, P19, or P25, in combination with Poncho™ (clothianidin)available from Bayer Crop Science, Poncho™ VOTiVO (clothianidin andBacillus firmus biological nematicide) available from Bayer CropScience, and Gaucho™ (imidicloprid) available from Bayer Crop Science.

Another example includes one or more of peptides of the presentinvention, including (without limitation) one of P1, P4-14S, P6a, P14d,P15a, P18, P19, or P25, in combination with Cruiser™ (thiamethoxam)available from Syngenta, CruiserMaxx™ (thiamethoxam, mefenoxam, andfludioxynil) available from Syngenta, Cruiser Extreme™ (thiamethoxam,mefenoxam, fludioxynil, and azoxystrobin) available from Syngenta,Avicta™ (thiamethoxam and abamectin) available from Syngenta, andAvicta™ Complete (thiamethoxam, abamectin, and Clariva Complete™ whichcontains the Pasteuria nishizawae—Pn1 biological inoculant) availablefrom Syngenta, and Avicta Complete™ Corn (thiamethoxam, mefenoxam,fludioxynil, azoxystrobin, thiabendazole and abamectin) available fromSyngenta.

Another example includes one or more of peptides of the presentinvention, including (without limitation) one of P1, P4-14S, P6a, P14d,P15a, P18, P19, or P25, in combination with Vault Liquid plus Integral(Bradyrhizobium species and Bacillus subtilis strain MBI 600 inoculants)available from BASF, Vault NP (Bradyrhizobium japonicum inoculant)available from BASF, and Subtilex NG (Bacillus subtilis biologicalinoculant) available from BASF.

The present invention further relates to methods of imparting diseaseresistance to plants, enhancing plant growth, effecting pest control,imparting biotic or abiotic stress tolerance to plants, and/ormodulating plant biochemical signaling. These methods involve applyingan effective amount of an isolated peptide of the invention, or acomposition of the invention to a plant or plant seed or the locus wherethe plant is growing or is expected to grow. As a consequence of suchapplication, the peptide contacts cells of the plant or plant seed, andinduces in the plant or a plant grown from the plant seed diseaseresistance, growth enhancement, tolerance to biotic stress, tolerance toabiotic stress, or altered biochemical signaling. Alternatively, thepeptide or composition of the invention can be applied to plants suchthat seeds recovered from such plants themselves are able to impartdisease resistance in plants, to enhance plant growth, to affect insectcontrol, to impart tolerance to biotic or abiotic stress, and/or tomodulate biochemical signaling, to modulate maturation.

In these embodiments, it is also possible to select plants or plantseeds or the locus to which the isolated peptide or composition of theinvention is applied. For example, for fields known to contain a highnematode content, the plants or plant seeds to be grown in such fields,or the fields (locus), can be selectively treated by applying theisolated peptide or composition of the invention as described herein;whereas no such treatment may be necessary for plants or plant seedsgrown in fields containing low nematode content. Similarly, for fieldshaving reduced irrigation, the plants or plant seeds to be grown in suchfields, or the fields (locus), can be selectively treated by applyingthe isolated peptide or composition of the invention as describedherein; whereas no such treatment may be necessary for plants or plantseeds grown in fields having adequate irrigation. Likewise, for fieldsprone to flooding, the plants or plant seeds to be grown in such fields,or the fields (locus), can be selectively treated by applying theisolated peptide or composition of the invention as described herein;whereas no such treatment may be necessary for plants or plant seedsgrown in fields that are not prone to flooding. As yet another exampleof such selection, for fields prone to insect attack at certain times ofthe growing season, the plants or plant seeds to be grown in suchfields, or the fields (locus), can be selectively treated by applyingthe isolated peptide or composition of the invention as describedherein; whereas the same field may not be treated at ineffective timesof the growing season or other fields that are not prone to such attackmay go untreated. Such selection steps can be carried out whenpracticing each of the methods of use described herein, i.e., impartingdisease resistance to plants, enhancing plant growth, effecting pestcontrol (including insects and nematodes), imparting biotic or abioticstress tolerance to plants, and/or modulating plant biochemicalsignaling.

As an alternative to applying an isolated peptide or a compositioncontaining the same to plants or plant seeds in order to impart diseaseresistance in plants, to effect plant growth, to control insects, toimpart stress resistance and/or modulated biochemical signaling to theplants or plants grown from the seeds, transgenic plants or plant seedscan be utilized. When utilizing transgenic plants, this involvesproviding a transgenic plant transformed with a DNA molecule encoding apeptide of the invention and growing the plant under conditionseffective to permit that DNA molecule to impart disease resistance toplants, to enhance plant growth, to control insects, to impart toleranceto biotic or abiotic stress, and/or to modulate biochemical signaling.Alternatively, a transgenic plant seed transformed with a DNA moleculeencoding a peptide of the invention can be provided and planted in soil.A plant is then propagated from the planted seed under conditionseffective to permit that DNA molecule to express the peptide and therebyimpart disease resistance to the transgenic plant, to enhance plantgrowth, to control insects, to impart tolerance to biotic or abioticstress, and/or to modulate biochemical signaling.

The present invention further relates to methods of improvingdesiccation resistance for cuttings removed from ornamental plants,post-harvest disease resistance or desiccation resistance to fruit orvegetables harvested from plants, and/or improved longevity of fruit orvegetable ripeness for fruit or vegetables harvested from plants. Thesemethods involve applying an effective amount of an isolated peptide ofthe present invention or a composition according to the presentinvention to a plant or the locus where the plant is growing. As aconsequence of such application, the peptide contacts cells of the plantor plant seed, and induces desiccation resistance for cuttings removedfrom ornamental plants, post-harvest disease resistance or desiccationresistance to fruit or vegetables harvested from plants, and/or improvedlongevity of fruit or vegetable ripeness for fruit or vegetablesharvested from plants. Alternatively, an effective amount of an isolatedpeptide of the present invention or a composition according to thepresent invention can be applied to a harvested fruit or vegetable. As aconsequence of such application, the peptide contacts cells of theharvested fruit or vegetable, and induces post-harvest diseaseresistance or desiccation resistance to the treated fruit or vegetables,and/or improved longevity of fruit or vegetable ripeness for the treatedfruit or vegetables.

As an alternative to applying an isolated peptide or a compositioncontaining the same to plants or plant seeds in order to inducedesiccation resistance to cuttings removed from ornamental plants,post-harvest disease resistance or desiccation resistance to fruit orvegetables harvested from plants, and/or improved longevity of fruit orvegetable ripeness for fruit or vegetables harvested from plants,transgenic plants or plant seeds can be utilized. When utilizingtransgenic plants, this involves providing a transgenic planttransformed with a DNA molecule encoding a peptide of the invention andgrowing the plant under conditions effective to permit that DNA moleculeto induce desiccation resistance for cuttings removed from ornamentalplants, post-harvest disease resistance or desiccation resistance tofruit or vegetables harvested from the transgenic plants, and/orimproved longevity of fruit or vegetable ripeness for fruit orvegetables harvested from the transgenic plants. Alternatively, atransgenic plant seed transformed with a DNA molecule encoding a peptideof the invention can be provided and planted in soil. A plant is thenpropagated from the planted seed under conditions effective to permitthat DNA molecule to express the peptide and thereby induce desiccationresistance for cuttings removed from ornamental plants, post-harvestdisease resistance or desiccation resistance to fruit or vegetablesharvested from the transgenic plants, and/or improved longevity of fruitor vegetable ripeness for fruit or vegetables harvested from thetransgenic plants.

In these embodiments, it is also possible to select transgenic plants orplant seeds for carrying out the present invention. For example, forfields known to contain a high nematode content, the transgenic plantsor plant seeds can be selectively grown in such fields; whereasnon-transgenic plants or plant seeds can be grown in fields containinglow nematode content. Similarly, for fields having reduced irrigation,the transgenic plants or plant seeds can be selectively grown in suchfields; whereas non-transgenic plants or plant seeds can be grown infields having adequate irrigation. Likewise, for fields prone toflooding, the transgenic plants or plant seeds can be grown in suchfields; whereas non-transgenic plants or plant seeds can be grown infields that are not prone to flooding. As yet another example of suchselection, for fields prone to insect attack at certain times of thegrowing season, the transgenic plants or plant seeds can be selectivelygrown in such fields; whereas non-transgenic plants or plant seeds canbe grown in fields that are not prone to such insect attack. Suchselection steps can be carried out when practicing each of the methodsof use described herein, i.e., imparting disease resistance to plants,enhancing plant growth, effecting pest control (including insects andnematodes), imparting biotic or abiotic stress tolerance to plants,and/or modulating plant biochemical signaling.

The present invention further relates to methods of improvingdesiccation resistance for cuttings removed from ornamental plants,post-harvest disease resistance or desiccation resistance to fruit orvegetables harvested from plants, and/or improved longevity of fruit orvegetable ripeness for fruit or vegetables harvested from plants. Thesemethods involve applying an effective amount of an isolated peptide ofthe present invention or a composition according to the presentinvention to a plant or the locus where the plant is growing. As aconsequence of such application, the peptide contacts cells of the plantor plant seed, and induces desiccation resistance for cuttings removedfrom ornamental plants, post-harvest disease resistance or desiccationresistance to fruit or vegetables harvested from plants, and/or improvedlongevity of fruit or vegetable ripeness for fruit or vegetablesharvested from plants. Alternatively, an effective amount of an isolatedpeptide of the present invention or a composition according to thepresent invention can be applied to a harvested fruit or vegetable. As aconsequence of such application, the peptide contacts cells of theharvested fruit or vegetable, and induces post-harvest diseaseresistance or desiccation resistance to the treated fruit or vegetables,and/or improved longevity of fruit or vegetable ripeness for the treatedfruit or vegetables.

In these embodiments, it is also possible to select plants, cuttings,fruits, vegetables, or the locus to which the isolated peptide orcomposition of the invention is applied. For example, for harvestedcuttings or fruit or vegetables that are being shipped great distancesor stored for long periods of time, then these can be selectivelytreated by applying the isolated peptide or composition of the inventionas described herein; whereas harvested cuttings or fruit or vegetablesthat are being shipped locally and intended to be consumed withoutsubstantially periods of storage can be excluded from such treatment.

As an alternative to applying an isolated peptide or a compositioncontaining the same to plants or plant seeds in order to inducedesiccation resistance to cuttings removed from ornamental plants,post-harvest disease resistance or desiccation resistance to fruit orvegetables harvested from plants, and/or improved longevity of fruit orvegetable ripeness for fruit or vegetables harvested from plants,transgenic plants or plant seeds can be utilized. When utilizingtransgenic plants, this involves providing a transgenic planttransformed with a DNA molecule encoding a peptide of the invention andgrowing the plant under conditions effective to permit that DNA moleculeto induce desiccation resistance for cuttings removed from ornamentalplants, post-harvest disease resistance or desiccation resistance tofruit or vegetables harvested from the transgenic plants, and/orimproved longevity of fruit or vegetable ripeness for fruit orvegetables harvested from the transgenic plants. Alternatively, atransgenic plant seed transformed with a DNA molecule encoding a peptideof the invention can be provided and planted in soil. A plant is thenpropagated from the planted seed under conditions effective to permitthat DNA molecule to express the peptide and thereby induce desiccationresistance for cuttings removed from ornamental plants, post-harvestdisease resistance or desiccation resistance to fruit or vegetablesharvested from the transgenic plants, and/or improved longevity of fruitor vegetable ripeness for fruit or vegetables harvested from thetransgenic plants.

In these embodiments, it is also possible to select transgenic plants orplant seeds for carrying out the present invention. For example,transgenic plants or plant seeds can be selected for growing when it isknown that harvested cuttings or fruit or vegetables are intended to beshipped great distances or stored for long periods of time post-harvest;whereas non-transgenic plants or plant seeds can be selected for growingwhen it is known that harvested cuttings or fruit or vegetables areintended to be shipped locally and/or consumed without substantiallyperiods of storage.

Suitable plants include dicots and monocots, including agricultural,silvicultural, ornamental and horticultural plants, whether in a naturalor genetically modified form. Exemplary plants include, withoutlimitation, alfalfa, apple, apricot, asparagus, avocados, bananas,barley, beans, beech (Fagus spec.), begonia, birch, blackberry,blueberry, cabbage, camphor, canola, carrot, castor oil plant, cherry,cinnamon, citrus, cocoa bean, coffee, corn, cotton, cucumber, cucurbit,eucalyptus, fir, flax, fodder beet, fuchsia, garlic, geranium, grapes,ground nut, hemp, hop, juneberry, juncea (Brassica juncea), jute,lentil, lettuce, linseed, melon, mustard, nectarine, oak, oats, oilpalm, oil-seed rape, olive, onion, paprika, pea, peach, pear,pelargonium, peppers, petunia, pine (Pinus spec.), plum, poplar (Populusspec.), pome fruit, potato, rape, raspberry, rice, rubber tree, rye,sorghum, soybean, spinach, spruce, squash, strawberry, sugar beet, sugarcane, sunflower, tea, teak, tobacco, tomato, triticale, turf,watermelon, wheat and willow (Salix spec.), Arabidopsis thaliana,Saintpaulia, poinsettia, chrysanthemum, carnation, and zinnia.

With respect to modified biochemical signaling, this includes bothenhancement of certain plant biochemical pathways and diminishment ofcertain other plant biochemical pathways. Biochemical signaling pathwaysthat can be altered in accordance with the present invention includegene expression and protein production, production of metabolites, andproduction of signaling molecules/secondary metabolites. Exemplarybiochemical signaling pathways and their modifications include, withoutlimitation, induction of nitric oxide production, peroxide production,and other secondary metabolites; agonist of the ethylene signalingpathway and induction of ethylene-responsive gene expression (see Donget al., Plant Phys. 136:3628-3638 (2004); Li et al., Planta 239:831-46(2014); Chang et al., PLoS One 10,e0125498 (2015), each of which ishereby incorporated by reference in its entirety); agonist of thesalicylic acid signaling pathway and induction of salicylicacid-responsive gene expression (see Dong et al., Plant J. 20:207-215(1999), which is hereby incorporated by reference in its entirety);agonist of the abscisic acid pathway and induction of abscisicacid-responsive gene expression (see Dong et al., Planta 221: 313-327(2005), which is hereby incorporated by reference in its entirety);agonist of the gibberellin signaling pathway and induction ofgibberellin-responsive gene expression (see Li et al., Planta 239:831-46(2014), which is hereby incorporated by reference in its entirety);antagonist of jasmonic acid signaling and inhibiting expression ofjasmonic acid-responsive genes (see Dong et al., Plant Phys.136:3628-3638 (2004), which is hereby incorporated by reference in itsentirety); inducing protease inhibitor expression (see Laluk andMengiste, Plant J. 68:480-494 (2011); Xia et al., Chin. Sci. Bull 56:2351-2358 (2011), each of which is hereby incorporated by reference inits entirety); inducing reactive oxygen species production in planttissues; inducing immune-related and antimicrobial peptide production,such as, without limitation, peroxidase, superoxide dismutase,chitinase, and β-1,3-glucanase (Wang et al., J. Agric. Food Chem.59:12527-12533 (2011), which is hereby incorporated by reference in itsentirety); and inducing expansin gene expression and production (see Liet al., Planta 239:831-46 (2014), which is hereby incorporated byreference in its entirety).

With respect to disease resistance, absolute immunity against infectionmay not be conferred, but the severity of the disease is reduced andsymptom development is delayed. Lesion number, lesion size, and extentof sporulation of fungal pathogens are all decreased. This method ofimparting disease resistance has the potential for treating previouslyuntreatable diseases, treating diseases systemically which might not betreated separately due to cost, and avoiding the use of infectiousagents or environmentally harmful materials.

The method of imparting pathogen resistance to plants in accordance withthe present invention is useful in imparting resistance to a widevariety of pathogens including viruses, bacteria, and fungi. Resistance,inter alia, to the following viruses can be achieved by the method ofthe present invention: Tobacco mosaic virus and Tomato mosaic virus.Resistance, inter alia, to the following bacteria can also be impartedto plants in accordance with present invention: pathogenic Pseudomonasspp., pathogenic Erwinia spp., pathogenic Xanthomonas spp., andpathogenic Ralstonia spp. Plants can be made resistant, inter alia, tothe following fungi by use of the method of the present invention:Fusarium spp. and Phytophthora spp.

With regard to the use of the peptides or compositions of the presentinvention to enhance plant growth, various forms of plant growthenhancement or promotion can be achieved. This can occur as early aswhen plant growth begins from seeds or later in the life of a plant. Forexample, plant growth according to the present invention encompassesgreater yield, increased plant vigor, increased vigor of seedlings(i.e., post-germination), increased plant weight, increased biomass,increased number of flowers per plant, higher grain and/or fruit yield,increased quantity of seeds produced, increased percentage of seedsgerminated, increased speed of germination, increased plant size,decreased plant height (for wheat), greater biomass, more and biggerfruit, earlier fruit coloration, earlier bud, fruit and plantmaturation, more tillers or side shoots, larger leaves, delayed leafsenescence, increased shoot growth, increased root growth, alteredroot/shoot allocation, increased protein content, increased oil content,increased carbohydrate content, increased pigment content, increasedchlorophyll content, increased total photosynthesis, increasedphotosynthesis efficiency, reduced respiration (lower O₂ usage),compensation for yield-reducing treatments, increased durability ofstems (and resistance to stem lodging), increased durability of roots(and resistance to root lodging), better plant growth in low lightconditions, and combinations thereof. As a result, the present inventionprovides significant economic benefit to growers. For example, earlygermination and early maturation permit crops to be grown in areas whereshort growing seasons would otherwise preclude their growth in thatlocale. Increased percentage of seed germination results in improvedcrop stands and more efficient seed use. Greater yield, increased size,and enhanced biomass production allow greater revenue generation from agiven plot of land.

With regard to the use of the peptides or compositions of the presentinvention to control pests (including but not limited to insects andnematodes, which are biotic stressors), such pest control encompassespreventing pests from contacting plants to which the peptide orcomposition of the invention has been applied, preventing direct damageto plants by feeding injury, causing pests to depart from such plants,killing pests proximate to such plants, interfering with insect larvalfeeding on such plants, preventing pests from colonizing host plants,preventing colonizing insects from releasing phytotoxins, interferingwith egg deposition on host plants, etc. The present invention alsoprevents subsequent disease damage to plants resulting from pestinfection.

The present invention is effective against a wide variety of insects(biotic stressors). European corn borer is a major pest of corn (dentand sweet corn) but also feeds on over 200 plant species includinggreen, wax, and lima beans and edible soybeans, peppers, potato, andtomato plus many weed species. Additional insect larval feeding pestswhich damage a wide variety of vegetable crops include the following:beet armyworm, cabbage looper, corn ear worm, fall armyworm, diamondbackmoth, cabbage root maggot, onion maggot, seed corn maggot, pickleworm(melonworm), pepper maggot, and tomato pinworm. Collectively, this groupof insect pests represents the most economically important group ofpests for vegetable production worldwide. The present invention is alsoeffective against nematodes, another class of economically importantbiotic stressors. Soybean Cyst Nematode (Heterodera glycines) is a majorpest of soybeans. Reniform Nematode (Rotylenchulus reniformis) is amajor pest of cotton as can parasitize additional crop species, notablysoy and corn. Additional nematode pests include the root knot nematodesof the genus Meloidogyne (particularly in cotton, wheat, and barley),cereal cyst nematodes of the genus Heterodera (particularly in soy,wheat, and barley), root lesion nematodes of the genus Pratylenchus,seed gall nematodes of the genus Anguina (particularly in wheat, barley,and rye), and stem nematodes of the genus Ditylenchus. Other bioticstressors include arachnids, weeds, and combinations thereof.

With regard to the use of the peptides or compositions of the presentinvention to impart abiotic stress resistance to plants, such abioticstress encompasses any environmental factor having an adverse effect onplant physiology and development. Examples of such environmental stressinclude climate-related stress (e.g., drought, flood, frost, coldtemperature, high temperature, excessive light, and insufficient light),air pollution stress (e.g., carbon dioxide, carbon monoxide, sulfurdioxide, NO_(x), hydrocarbons, ozone, ultraviolet radiation, acidicrain), chemical (e.g., insecticides, fungicides, herbicides, heavymetals), nutritional stress (e.g., over- or under-abundance offertilizer, micronutrients, macronutrients, particularly potassium,nitrogen derivatives, and phosphorus derivatives), and improved healingresponse to wounding. Use of peptides of the present invention impartsresistance to plants against such forms of environmental stress.

A further aspect of the present invention relates to the use of thepeptides of the present invention as a safener in combination with oneor more of the active agents (i.e., in a composition or in separatecompositions) for the control of aquatic weeds in a body of water asdescribed in U.S. Publ. No. 20150218099 to Mann, which is herebyincorporated by reference in its entirety.

Yet another aspect of the present invention relates to the use of thepeptides of the present invention as a plant strengthener in acomposition for application to plants grown under conditions of reducedwater irrigation, which composition also includes at least oneantioxidant and at least one radiation manager, and optionally at leastone plant growth regulator, as described in U.S. Publ. No. 20130116119to Rees et al., which is hereby incorporated by reference in itsentirety.

The methods of the present invention involving application of thepeptide or composition can be carried out through a variety ofprocedures when all or part of the plant is treated, including leaves,stems, roots, propagules (e.g., cuttings), fruit, etc. This may (butneed not) involve infiltration of the peptide into the plant. Suitableapplication methods include high or low pressure spraying, injection,and leaf abrasion proximate to when peptide application takes place.When treating plant seeds, in accordance with the application embodimentof the present invention, the hypersensitive response elicitor proteinor polypeptide can be applied by low or high pressure spraying, coating,immersion (e.g., soaking), or injection. Other suitable applicationprocedures can be envisioned by those skilled in the art provided theyare able to effect contact of the hypersensitive response elicitorpolypeptide or protein with cells of the plant or plant seed. Oncetreated with the peptides or compositions of the present invention, theseeds can be planted in natural or artificial soil and cultivated usingconventional procedures to produce plants. After plants have beenpropagated from seeds treated in accordance with the present invention,the plants may be treated with one or more applications of the peptidesor compositions of the invention to impart disease resistance to plants,to enhance plant growth, to control insects on the plants, to impartbiotic or abiotic stress tolerance, to improve desiccation resistance ofremoved cuttings, to impart post-harvest disease resistance ordesiccation resistance to harvested fruit or vegetables, and/or improvedlongevity of fruit or vegetable ripeness for harvested fruit orvegetables.

The peptides or compositions of the invention can be applied to plantsor plant seeds in accordance with the present invention alone or in amixture with other materials. Alternatively, the peptides orcompositions can be applied separately to plants with other materialsbeing applied at different times.

In the alternative embodiment of the present invention involving the useof transgenic plants and transgenic seeds, a peptide of the inventionneed not be applied topically to the plants or seeds. Instead,transgenic plants transformed with a DNA molecule encoding a peptide ofthe invention are produced according to procedures well known in theart. A vector suitable for expression in plants (i.e., containingtranslation and transcription control sequences operable in plants) canbe microinjected directly into plant cells by use of micropipettes totransfer mechanically the recombinant DNA. Crossway, Mol. Gen. Genetics,202:179-85 (1985), which is hereby incorporated by reference in itsentirety. The genetic material may also be transferred into the plantcell using polyethylene glycol. Krens, et al., Nature, 296:72-74 (1982),which is hereby incorporated by reference in its entirety.

Another approach to transforming plant cells with a gene encoding thepeptide of the invention is particle bombardment (also known asbiolistic transformation) of the host cell. This can be accomplished inone of several ways. The first involves propelling inert or biologicallyactive particles at cells. This technique is disclosed in U.S. Pat. Nos.4,945,050, 5,036,006, and 5,100,792, all to Sanford et al., which arehereby incorporated by reference. Generally, this procedure involvespropelling inert or biologically active particles at the cells underconditions effective to penetrate the outer surface of the cell and tobe incorporated within the interior thereof. When inert particles areutilized, the vector can be introduced into the cell by coating theparticles with the vector containing the heterologous DNA.Alternatively, the target cell can be surrounded by the vector so thatthe vector is carried into the cell by the wake of the particle.Biologically active particles (e.g., dried bacterial cells containingthe vector and heterologous DNA) can also be propelled into plant cells.

Yet another method of introduction is fusion of protoplasts with otherentities, either minicells, cells, lysosomes or other fusiblelipid-surfaced bodies. Fraley, et al., Proc. Natl. Acad. Sci. USA,79:1859-63 (1982), which is hereby incorporated by reference in itsentirety. The DNA molecule may also be introduced into the plant cellsby electroporation. Fromm et al., Proc. Natl. Acad. Sci. USA, 82:5824(1985), which is hereby incorporated by reference in its entirety. Inthis technique, plant protoplasts are electroporated in the presence ofplasmids containing the expression cassette. Electrical impulses of highfield strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and regenerate.

Another method of introducing the DNA molecule into plant cells is toinfect a plant cell with Agrobacterium tumefaciens or A. rhizogenespreviously transformed with the gene. Under appropriate conditions knownin the art, the transformed plant cells are grown to form shoots orroots, and develop further into plants. Generally, this procedureinvolves inoculating the plant tissue with a suspension of bacteria andincubating the tissue for 48 to 72 hours on regeneration medium withoutantibiotics at 25-28° C. Agrobacterium is a representative genus of thegram-negative family Rhizobiaceae. Its species are responsible for crowngall (A. tumefaciens) and hairy root disease (A. rhizogenes). The plantcells in crown gall tumors and hairy roots are induced to produce aminoacid derivatives known as opines, which are catabolized only by thebacteria. The bacterial genes responsible for expression of opines are aconvenient source of control elements for chimeric expression cassettes.In addition, assaying for the presence of opines can be used to identifytransformed tissue. Heterologous genetic sequences can be introducedinto appropriate plant cells, by means of the Ti plasmid of A.tumefaciens or the Ri plasmid of A. rhizogenes. The Ti or Ri plasmid istransmitted to plant cells on infection by Agrobacterium and is stablyintegrated into the plant genome. J. Schell, Science, 237:1176-83(1987), which is hereby incorporated by reference in its entirety.

After transformation, the transformed plant cells must be regenerated.Plant regeneration from cultured protoplasts is described in Evans etal., Handbook of Plant Cell Cultures, Vol. 1: (MacMillan Publishing Co.,New York, 1983); and Nasil I.R. (ed.), Cell Culture and Somatic CellGenetics of Plants, Acad. Press, Orlando, Vol. 1, 1984, and Vol. III(1986), which are hereby incorporated by reference in their entirety.

It is known that practically all plants can be regenerated from culturedcells or tissues. Means for regeneration vary from species to species ofplants, but generally a suspension of transformed protoplasts or a petriplate containing transformed explants is first provided. Callus tissueis formed and shoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced in the callus tissue.These embryos germinate as natural embryos to form plants. The culturemedia will generally contain various amino acids and hormones, such asauxin and cytokinins It is also advantageous to add glutamic acid andproline to the medium, especially for such species as corn and alfalfa.Efficient regeneration will depend on the medium, on the genotype, andon the history of the culture. If these three variables are controlled,then regeneration is usually reproducible and repeatable.

After the expression cassette is stably incorporated in transgenicplants, it can be transferred to other plants by sexual crossing. Any ofa number of standard breeding techniques can be used, depending upon thespecies to be crossed.

Once transgenic plants of this type are produced, the plants themselvescan be cultivated in accordance with conventional procedure with thepresence of the gene encoding the hypersensitive response elicitorresulting in disease resistance, enhanced plant growth, control ofinsects on the plant, abiotic or biotic stress tolerance, improveddesiccation resistance of removed cuttings, post-harvest diseaseresistance or desiccation resistance in harvested fruit or vegetables,and/or improved longevity of fruit or vegetable ripeness for harvestedfruit or vegetables.

Alternatively, transgenic seeds are recovered from the transgenicplants. These seeds can then be planted in the soil and cultivated usingconventional procedures to produce transgenic plants. The transgenicplants are propagated from the planted transgenic seeds under conditionseffective to impart disease resistance to plants, to enhance plantgrowth, to control insects, to impart abiotic or biotic stresstolerance, to improve desiccation resistance of removed cuttings, toimpart post-harvest disease resistance or desiccation resistance inharvested fruit or vegetables, and/or to impart improved longevity offruit or vegetable ripeness for harvested fruit or vegetables.

When transgenic plants and plant seeds are used in accordance with thepresent invention, they additionally can be treated with the samematerials as are used to treat the plants and seeds to which a peptideof the invention or composition of the invention is applied. These othermaterials, including peptides or composition of the invention, can beapplied to the transgenic plants and plant seeds by the above-notedprocedures, including high or low pressure spraying, injection, coating,and immersion. Similarly, after plants have been propagated from thetransgenic plant seeds, the plants may be treated with one or moreapplications of the peptides or compositions of the invention to impartdisease resistance, enhance growth, control insects, abiotic or bioticstress tolerance, desiccation resistance of removed cuttings,post-harvest disease resistance or desiccation resistance in harvestedfruit or vegetables, and/or improved longevity of fruit or vegetableripeness for harvested fruit or vegetables.

Such transgenic plants may also be treated with conventional planttreatment agents, e.g., bacteriocidal or biocidal agents, proteaseinhibitors, non-ionic surfactants, fertilizers, herbicides,insecticides, fungicides, nematicides, biological inoculants, plantregulators, and mixtures thereof, as described above.

EXAMPLES

The following examples are provided to illustrate embodiments of thepresent invention but are by no means intended to limit its scope.

Example 1 Development of “HR Box” Peptides of SEQ ID NO: 93

The HR box was originally developed based on examination of a number ofHypersensitive Response-Inducing sequences (P1, SEQ ID NO: 4; P4, SEQ IDNO: 5; and P15, SEQ ID NO: 64, among others). A repeating sequence ofleucine and isoleucine residues was identified. P4 was chosen as arepresentative sequence as the basis for mutational studies that wouldreveal the sequence determinants of HR elicitation. HR in tobacco wastested as described in Wei, Science 257:85-88 (1992), which is herebyincorporated by reference in its entirety. Briefly, peptides weredissolved at a concentration of 500 μg/ml in aqueous solution. Fourserial dilutions were performed with an equal volume of water, yieldingpeptide samples at 500, 250, 125, 62.5, 31.25 μg/ml peptide solutions.Nicotiana tabacum cultivar xanthi plants were used at 5-7 weeks old(preflowering). Leaves were lightly punctured with a toothpick in amiddle leaf panel. Peptide solutions were then infused via needle-lesssyringe into the wound, filling the panel. Each peptide sample wasinfused into a leaf of 2 different plants. The leaves were observed andscored over the next 48 hours for withering and browning, lesionstypical of programmed cell death. These mutational studies had threemain goals: (1) increase the solution stability of the peptides; (2)make disruptive mutations to verify the residues which are mostimportant for HR elicitation; and (3) make conservative mutations toidentify the degree of specificity for particular amino acids.

Peptides were assessed for one or more of solubility, stability againstchemical degradation, effect of bulking agents on solution stability,oxidation protection, and solution stability studies.

Solubility was assessed by creating 0.2% AI (active ingredient)solutions of pure, chemically synthesized peptide in deionized water,and observing the solution for evidence of precipitation over 48 hoursat room temperature. P1 (SEQ ID NO: 4) was largely insoluble in water.However, the mutant with several glutamine residues replacing glutamateresidues (P1-2E,8E,11E,15E,18E, SEQ ID NO: 46) was soluble. P4 (SEQ IDNO: 5) and P4-14S (SEQ ID NO: 6) were also completely soluble.

Subsequent experiments were run to better quantify peptide solubility.20-50 mg of pure peptide were mixed with 0.25 ml of water and increasingamounts of water were added until the peptide dissolved. Theseexperiments estimate the solubility of P1 (SEQ ID NO: 4) at <1 mg/ml,the solubility of P4 (SEQ ID NO: 5) at 100 mg/ml, and the solubility ofP1-18K (SEQ ID NO: 45) at 20 mg/ml.

Stability against chemical degradation was assessed in various pHbuffers by creating 0.2% AI solutions of pure, chemically synthesizedpeptide in deionized water, 0.25% weight to volume of Proxel® GXL(biocide), and 50 millimolar (mM) of eight buffers (separately) asfollows: MES pH 5.6, MOPS pH 6.5, Citrate pH 7.2, EDDS pH 7.3, EDTA pH8, Phosphate pH 8, Imidazole pH 8, and TES pH 8. The solutions wereobserved on HPLC for evidence of degradation (% loss of the peptidesignal over time, relative to the time 0 sample) over a period of weeksat elevated temperature (50° C.). P1-2E,-8E,-11E,-15E,-18E (SEQ ID NO:46) was more stable than P1 (SEQ ID NO: 4) (40 days vs 20 days over80%), and P4-14S (SEQ ID NO: 6) was significantly more stable than P4(SEQ ID NO: 5) (35 days vs 3 days over 80%). The best buffers for P1 andP4-14S are TES pH 8 and Citrate pH 7.2, in that order. Precipitation ofP1 was observed after several days. Other peptides(P1-2E,-8E,-11E,-15E,-18E; P4, and P4-14s) remained in solution.

Effect of bulking agents on the chemical degradation of P1 andP1-2E,-8E,-11E,-15E,-18E was assessed by creating 0.2% AI solutions ofpure, chemically synthesized peptide in a solution of 50 mM TES pH 8.0in water and 20% weight to volume (of solution) of the bulking agentstrehalose, maltrin, sucrose or talc (separate formulas). These solutionswere observed on HPLC for evidence of degradation (% loss of the peptidesignal over time, relative to the time 0 sample) over time at elevatedtemperature (50° C.). The concentration of P1 in all mixtures dropped toless than 60% of the original peptide concentration after 6 days ofincubation. In contrast, the concentration of p1-2E,-8E,-11E,-15E,-18Ein all samples remained above 80% of the original concentration for atleast 14 days. The best bulking agent for P1-2E,-8E,-11E,-15E,-18E istalc powder (44 days above 80%).

Solution stability studies were carried out by creating 0.2% AIsolutions of pure, chemically synthesized peptide in deionized water, 50mM of TES buffer, 0.25% Proxel GXL, and 30% isopropanol. Peptidessolutions were analyzed by HPLC for % loss of the peptide signal overtime, relative to the time 0 sample. Maximum lifetime ofP1-2E,-8E,-11E,-15E,-18E is 45 days over 80%. Maximum lifetime of P4-14Sis 140 days over 80%.

Solution stability mutations: Solution stability was increased bychoosing a peptide sequence (P4, SEQ ID NO: 5) which did not containmethionine residues. However, this peptide contained a cysteine residue,leading to very poor stability. Mutation of this cysteine to theconservative replacement serine (sulfur to oxygen change in chemicalstructure) generated P4-14s (SEQ ID NO: 6), which retained its abilityto elicit the HR. It was subsequently shown (as noted above) that P4-14sis a highly stable peptide. Additional studies replaced one or moreglutamine residues with glutamate residues to reduce the chance ofdeamidation in solution. In particular, a variant of P1, termed P1-2E,8E, 11E, 15E, 18E (SEQ ID NO: 46), contained these mutations atpositions 2, 8, 11, 15, and 18. This peptide exhibited both improvedsolubility and stability when compared with P1.

Based on the P4-14s stable backbone (SEQ ID NO: 6), disruptive singlemutations were introduced at specific residues within the sequence. Inthe case of Leucine residues, these were mutated to alanine (smaller andless hydrophobic sidechain, a moderately disruptive mutation) and/oraspartic acid (negatively charged sidechain, a highly disruptivemutation). The intervening sequences, depending on the identity of theamino acid in question, were mutated to have a negative charge (asparticacid or glutamic acid), have a hydrophobic sidechain (valine), a minimalsidechain (alanine), or a small polar sidechain (serine). These mutantpeptides were tested for elicitation of the hypersensitive response.Additional mutations were chosen based on the initial HR results. Inaddition, the spacing between the leucine/isoleucine residues wasevaluated by either deleting a single residue between the leucinerepeats (denoted with ‘del’) or by inserting an alanine residue betweenthe leucine repeats (denoted with iA).

For those amino acids that were important for HR elicitation, moreconservative mutations were chosen to determine the specificity ofinteractions. The leucine residues were mutated to isoleucine, valine,phenylalanine or tyrosine, with the latter 2 residues being lessconservative. As above, these mutants were tested for HR elicitation.

The results of these mutation studies are summarized in Table 12 below:

TABLE 12 Summary of Mutations and HR Elicitation Results 1 2 3 4 5 6 7 89 10 11 12 13 14 15 16 17 18 19 20 21 22 23 P4-14S S Q G I S E K Q L D QL L S Q L I Q A L L Q P HR Positive E E L V R V D E iA del V I I S E I ME D I F V Mutations dN2 A dN5 dN6 V V I V V M T A M V S S M I dC2 D S VM A I F L M V M dN4 F V K T D S K K V V Q del Weak HR F V A A VMutations S S HR A iA D A iA A D del S D Negative D D S S iA V MutationsY V F F S F Q

In Table 12, the sequence of P4-14s is shown along with all mutationstested at each position. Those mutations that did not interfere with thehypersensitive response are listed in the labeled “HR PositiveMutations”. Those mutations that caused a reduction in the severity ofthe hypersensitive response are shown in the row labeled “Weak HRMutations”. Mutations that eliminated the hypersensitive response areshown in the red row labeled “HR Negative Mutations”. The notation dN2and dN4 denote a deletion of 2 or 4 residues, respectively, from thebeginning of the peptide; dC2 denotes a deletion of 2 residues from theend of the peptide; del denotes a deletion of the residue at thatposition; and iA represents the insertion of an alanine residue beforethat position.

Example 2 Solubility and Stability of P1 and Mutant Peptides

As described above, P1 and P1-derived sequences mutated at position 18(methionine replaced with alanine, threonine, or lysine) were assessedfor solution stability and chemical compatibility for 14 days. Notably,P1 exhibited solubility problems at lower pH (in deionized watersolution, in 50 mM citrate pH 5.6, and in 50 mM MES pH 6.0). In thesecases, the peptide concentration increased after 24-hour incubation at50° C. Notably, the mutant peptides generally did not exhibit thisissue. As shown in FIGS. 1-3, data were normalized to 100% peptide atthe day 1 time point (shown as peptide 1* in the legend and the originalpeptide 1 data is marked with a double asterisk **). In a stability testdissolved in water (FIG. 1), peptide 1 was modestly stable, butexhibited solubility issues. The 18K and 18A mutants exhibited slightlyhigher stability (10-25% after 14 days). Dissolved in a slightly acidiccitrate buffer (FIG. 2), P1 exhibited both solubility and stabilityissues. It was not detected by HPLC after 14 days in solution. Bycontrast the 18T and 18K mutants retained 80% of the originalconcentration, and the 18A mutant retained ˜60% of the originalconcentration. As shown in FIG. 3, in 50 mM MES pH 6.0, P1 exhibitedstronger solubility problems, with a 50% increase in solubleconcentration after 24 hour incubation at 50° C. However, it exhibitedbetter stability than the mutants (10-30% after 14 days). In citrate pH7.2 (FIG. 6), P1 did not exhibit solubility problems, but did exhibitpoor stability (20% of original concentration after 7 days at 50° C. Bycontrast, the 18K and 18T mutants exhibited >60% stability after 14days. In 50 mM EDDS, pH 7.3 (FIG. 7), Peptide 1 exhibited poorstability, with only 10% of the material remaining after 7 days. Bycomparison, the mutants retained at least 50% of starting material after14 days. In 50 mM imidazole, pH 8.0 (FIG. 8), Peptide 1 exhibitedparticularly poor stability, reduced to less that 10% of the originalconcentration after only 3 days. By comparison, all mutants exhibitedgreater stability, with 18K and 18T mutants retaining 60-75% of theoriginal material after 14 days. Peptide 1 exhibited better stability ina solution of 50 mM EDTA, pH 8.0 (FIG. 9), matching the performance ofthe 18A mutant. However, the 18K and 18T mutants exhibited bettertability after 14 days incubated at 50° C. (15-20% improvement). Whendissolved in phosphate, pH 8.0 (FIG. 10), Peptide 1 appears tooutperform the stability of the mutants, although it does exhibitsolubility problems (some cloudiness in solution). In a solution of 50mM TES, pH 8.0 (FIG. 11), Peptide 1 is more than 90% degraded after 1week of incubation at 80° C. By comparison, 18T, 18K, and 18A mutantsexhibit better performance (71%, 58%, and 47% remaining after 14 daysincubation at 50° C.).

In general, Peptide 1 either exhibits solubility problems or poorstability in a wide variety of buffered solutions. This is addressed bymutation of methionine to other residues. Bulkier residues (threonineand lysine) generally seem preferred over alanine for stability.

Example 3 Solubility and Stability of P4 and Mutant Peptides

As described above, P4 and P4-derived sequences mutated at position 14(cysteine replaced with alanine/A, aspartic acid/D, lysine/K,glutamine/Q, and serine/S) were assessed for solution stability andchemical compatibility for 14 days. In general, peptide 4 exhibited verypoor stability due to the presence of cysteine (FIGS. 12-21). After lessthan 1 day, the original P4 HPLC peak was not detected in the samples.By comparison, all mutants exhibited better stability. Most of theseretained at least 50% of the original material for 14 days at 50° C. Ingeneral, peptide 4 mutants exhibit better stability at higher pH values(>7.0). Notably, p4-14s can regularly exhibit stability of 90% after 14days, depending on conditions. All of the mutant peptides exhibited thehypersensitive response when infiltrated into tobacco leaves (as inexample 1).

Example 4 Comparison of Peptide 1 and Peptide 4 Stability

Although peptide 1 and peptide 4 exhibit a high degree of sequencesimilarity, the stabilized mutants of peptide 4 are more stable than thepl mutants. A series of mutations of p1 were made to confer stabilitysimilar to p4-14s. These are: p1-1S (SEQ ID NO: 109, Table 1), p1-14S(SEQ ID NO: 110, Table 1), p1-18Q (SEQ ID NO: 115, Table 1), p1-23P (SEQID NO: 118, Table 1). These peptides, along with p1 and p4-14s, weredissolved in 30% isopropanol, 5 mM DTPA, and 50 mM TES pH 8.0 and testedfor stability at 50 C. Similar stability was observed for p4-14S andp1-1S, indicating that the N-terminal amino acid exhibits a strongeffect on peptide stability.

Example 5 Solubility for P15b and Mutants

Initial results suggest that pl5b has solubility problems. It has arelatively high hydrophobicity (0.19). At 0.2% w/v, it was partiallysoluble in water, insoluble in 50 mM of each of citrate pH ˜5.2, citratepH 7.0, phosphate pH 7.0 (check), TES pH 8.0, EDTA pH 8.0, and EDDS pH7.0. It was at least partially soluble in 50 mM MES pH 6.0, and MOPS pH6.5. However, P15a dissolves more easily in aqueous solutions. Itssolubility is >10 mg/ml in 50 mM TES pH 8.0. Additional p15 variantswere synthesized that included poly-glutamate solubility tags (p15-59Gand p15-59, SEQ ID NOS: 149 and 150, respectively). When p15-59 wasdissolved in 50 mM TES, pH 8.0, it exhibited solubility >10 mg/ml (1%w/v).

Example 6 Stability of P17/P18 & Variants

As described above, P18 (SEQ ID NO: 83) was tested for stability andchemical compatibility with different pH buffers. P18 exhibitsrelatively poor stability in aqueous buffer solutions at 50° C. Mostsamples degraded to 20% of original concentration in 3 days. Oneexception is a 50 mM EDTA solution, which degrades to 35% after 7 days(FIG. 24). Mutation of the methionine at position 12 to leucine (in P18-4, SEQ ID NO: 164) causes moderate stabilization: 60% stability after14 days. Notably, truncation of the last 3 amino acids from theC-terminus (P18-1, SEQ ID NO: 163) also leads to dramatically increasedstability (>90% stability over the 14-day trial).

Example 7 Stability of P19 & Variants

In general, P19 (SEQ ID NO: 89) exhibits relatively high stability, >80%stability over 14 days at 50° C. under a variety of conditions. Theexceptions were peptide dissolved in water alone (52%) or 50 mM TES pH8.0 (62%). Mutation of its one methionine residue at position 12 toleucine (P19-20L, SEQ ID NO: 90) leads to a modest increase in stabilitywhen dissolved in 50 mM citrate pH 7.2 or 50 mM TES, pH 8.0 (FIGS. 25and 26). When using buffers at lower pH (5.5-7.0), the performance ofP19 and P19-20L was observed to be similar; more than 80% peptide wasretained for 14 days.

Example 8 P14d, P14e, P14f Stability

P14d sequence (SEQ ID NO: 175) is derived from the popA sequence ofRalstonia solanacearum. It conforms to the HR-box motif and causes HR intobacco leaves. Mutation of the methionine residues generated thestabilized peptides P14e (SEQ ID NO: 176) and P14f (SEQ ID NO: 177). Themutated peptides exhibit >85% stability for >50 days at 50° C. (in 50 mMTES, pH 8.0 and 30% isopropanol). During the same period of time, P14dexhibits around 50% chemical stability.

Example 9 Growth Tests

For growth tests, corn and soy seeds were planted in flats with 2 seedsper cell within the flat at a greenhouse facility. The seeds wereallowed to germinate and the smaller plant is culled, leaving one plantper cell. Once the first true leaves are fully expanded and the secondleaves are beginning to expand, the plants were initially measured forheight. This was performed by stretching the highest leaf upward andmeasuring the distance to the soil. Peptides were dissolved in water atthe indicated concentrations (below). The plants were then treated witha foliar spray using widely available spray bottles until liquid wasdripping from the leaves. 4 flats of 14 plants each were treated percondition (peptide or control). Corn and soy were treated as indicatedin Table 13 and compared with matched water-treated control plants. Theplants were allowed to grow for 14 days. The height of the plants wasagain measured and compared to the original height to quantify growth.In some cases, the plants were allowed to grow without watering for 2-4days until the onset of wilting and drought stress. At this time, theabove-ground portions of the plants were harvested and weighed todetermine the fresh mass. Finally, the above-ground material was driedat 70° C. for 48 hours and weighed to determine dry biomass. The resultsof these growth trials are shown in Table 13. Growth, dry biomass, andfresh mass are calculated as the % increase over the water-treatedcontrol.

TABLE 13 Growth Trial Results SEQ ID Growth Dry biomass Fresh massPeptide NO: Rate Host (% difference) (% difference) (% difference) P15a63 0.2 Corn 6.0 5.0 N.D. P15b 49 0.2 Corn 0.9 2.0 13.9 P18 83 0.2 Corn15.0 9.0 N.D. P18 83 0.2 Soy 18.0 15.0 N.D. P19 89 0.2 Soy 18.0 12.6N.D. P25 182 5.0 Corn 7.0 11.0 N.D. P25 182 0.2 Soy 10.0 2.0 N.D.P4-14s-18E 191 2.0 Soy 9.0 0.8 N.D. P30-3 190 0.2 Corn 8.7 8.0 13.4P25-11 188 0.2 Corn 4.1 4.7  1.6 P25-11 188 5 Corn 1.0 6.2 10.9 N.D. =Not determined.

Several of the tested peptides exhibit growth and/or biomass increasesin corn and soy. Notably, although Pl5b did not cause an overt growth ordry biomass phenotype, it did cause an increase in fresh biomass, whichis suggestive of increased water uptake or retention. This is anindication of drought tolerance in those treated plants. Anotherpeptide, P30-3, was observed to cause an increase in growth, fresh mass,and dry biomass.

Example 10 Minimal Sequences Required for HR Response

After determining the residues most critical for hypersensitive responseelicitation, we designed additional mutants to verify the smallestpeptide sequence responsible for this behavior. Due to the hydrophobicnature of the core HR sequence (containing 7 leucine or isoleucineresidues in 13 residues total), it was recognized that solubility wouldbe a problem for the minimal peptide. As a result, hydrophilic sequenceswere added to many peptides to bring the hydrophobicity on theKyte-Doolittle scale to around −0.2 for the peptides.

Initially, a poly-lysine or poly-arginine sequence was used, i.e., P4having an N-linked polyR or polyK sequence, or a C-linked polyR or polyKsequence (SEQ ID NOS: 35, 36, 38, 39). However, when infiltrated intotobacco leaves, these peptides caused necrotic lesions not typical ofHR. This led to the hypothesis that poly-cationic sequences cause atoxic response when infiltrated into tobacco leaves. When poly-lysineand poly-arginine alone was infiltrated into the leaf, a similarnecrotic lesion was observed. As a result, testing of peptidescontaining cationic solubility enhancing sequences was discontinued.Notably, HR+ peptides can contain at least one or two cationic aminoacids, but larger numbers of positive charges appear to be detrimental.As a substitute for cationic peptides, polyanions were considered,specifically poly-glutamate. Poly-glutamate was chosen since aspartatehas a greater chance of isomerizing to iso-aspartate, and serine wasadded at the N-terminus to eliminate the formation of pyroglutamic acidat the N-terminus of the peptide. It is also reasonable to add glutamateresidues at the C-terminal end of the peptide.

The hypersensitive response test was run as described in example #1. ForP4, the smallest variant peptide that elicited HR was P4-polyE-min3 (SEQID NO: 33). For P1, the smallest variant peptide that elicited HR wasP1-polyE-min3 (SEQ ID NO: 141). For P18, the smallest variant peptidethat elicited HR was P18-7 (SEQ ID NO: 167). For P19, the smallestvariant peptide that elicited HR was P19-8 (SEQ ID NO: 173). For P15,the smallest variant peptide that elicited HR was P15-59 (SEQ ID NO:150). For P14d, the smallest variant peptide that elicited HR was P14-30(SEQ ID NO: 178). For P25, the smallest variant peptide that elicited HRwas P25-11 (SEQ ID NO: 188). In addition, minimal peptide sequences weregenerated incorporating the leucine repeat sequence characteristic ofthe HR-box and glutamic acid residues in the variable positions toincrease solubility. These sequences are: P30-2 (SEELEELLEELIEELL, SEQID NO: 189), P30-3 (LEELLEELIEELLEE, SEQ ID NO: 190), and P30-4(LEELLEELIEELL, SEQ ID NO: 210). These minimal HR-box sequences weresoluble >5 mg/ml in 50 mM TES and produced an HR response wheninfiltrated into tobacco leaves.

Likewise, additional peptides were developed for enhanced solubilitybased on the hydrophobic backbone sequences of P3, P25, P14, P15, andP19. These are listed in Table 10, supra.

Based on the previously described behavior of harpins and HR+ peptides,it is expected that these new peptides will have wide-rangingbioactivity including inducing resistance to TMV, resistance tonematodes, increased stress and drought resistance, increased growth,and increased yield as described in PCT Application WO 01/98501 to Fanet al., which is hereby incorporated by reference in its entirety.

Example 11 Derivatives of Peptide P1 that Cause HR Response in Tobacco

HR tests (described in Example 1) were run on variants of P1 todetermine the minimal sequence required for HR and to identify residuesof importance. The following peptides of Table 14 were determined to bepositive for HR:

TABLE 14 Peptide SEQ Name Sequence ID NO: P1 NQGISEKQLDQLLTQLIMALLQQ   4P1-allE, NEGISEKELDELLTELIEALLQQ  46 P1-18T NQGISEKQLDQLLTQLITALLQQ  42P1-18E NQGISEKQLDQLLTQLIEALLQQ  43 P1-18A NQGISEKQLDQLLTQLIAALLQQ  44P1-18K NQGISEKQLDQLLTQLIKALLQQ  45 P1-1S SQGISEKQLDQLLTQLIMALLQQ 109P1-14S NQGISEKQLDQLLSQLIMALLQQ 110 P1-18Q NQGISEKQLDQLLTQLIQALLQQ 115P1-23P NQGISEKQLDQLLTQLIMALLQP 118 polyE-min3p1   SEEEEELDQLLTQLIEALL141

Example 12 Derivatives of Peptide P3 that Cause HR Response in Tobacco

HR tests (described in Example 1) were run on variants of P3 todetermine the minimal sequence required for HR and to identify residuesof importance. The following peptides of Table 15 were determined to bepositive for HR.

TABLE 15 Peptide SEQ Name Sequence ID NO: P3QNDDSTSGTDSTSDSSDPMQQLLKMFSEIMQSLFGDGQDGT 204 P3-3               SDPMQQLLKMFSEIMQSLF 205 P3-4              SEEELQQLLKLFSEILQSLF 206 P3-6            SEEEEELQQLLKLFSEILQSL 207 P3-7            SEEEEELQQLLKLFSEILQS 208

It is notable that P3 seems to require a longer sequence than theminimal HR-box repeat for efficient HR elicitation. This may be due tothe sub-optimal phenylalanine residues and the presence of only a singleK residue to separate the hydrophobic residues (LLKLF in P3 and itsvariants) present in this sequence. However, it is important to notethat additional hydrophobic residues are not strictly necessary,considering that P3-6 and P3-7 cause HR.

Example 13 Derivatives of Peptide P25 that Cause HR Response in Tobacco

HR tests (described in Example 1) were run on variants of P25 todetermine the minimal sequence required for HR and to identify residuesof importance. The following peptides of Table 16 were determined to bepositive for HR.

TABLE 16 Peptide SEQ Name Sequence ID NO: P25     GGLTLTGVLQKLMKILNAL182 P25s  EDQGGLTLTGVLQKLMKILNAL 183 P25-4  EDQGGLTLTGVLQKLMKILNALVQ 181P25-10 SEEEEELTLTGVLQKLLKILEAL 187 P25-11 SEEEEELTGVLQKLLKILEAL 188P25-15 SEEEEELTLTGVLQKLLKILEA 200 P25-16      SEEEEEVLQKLLKILEALV 201P25-17       SEEEEELQKLLKILEALVQ 202

It is important to note that P25 variants seem to require more sequencethan the minimal HR consensus (SEQ ID NO:93) to HR elicitation. This maybe due to the presence of valine residues where leucine is preferred ordue to the presence of a single hydrophilic residue between thehydrophobic repeats (LLKIL). Although we include P25-15, P25-16, andP25-17 as HR+, they exhibited a very weak hypersensitive response thatonly occurred in some tobacco plants at the highest application rate.Notably, the additional sequence content does not seem to requireleucine/isoleucine/valine residues, as suggested by the biologicalresponse to P25-15.

Example 14 Derivatives of Peptide P14d that Cause HR Response in Tobacco

HR tests (described in Example 1) were run on variants of P14 todetermine the minimal sequence required for HR and to identify residuesof importance. The following peptides of Table 17 were determined to bepositive for HR.

TABLE 17 Peptide SEQ Name Sequence ID NO: P14d                  QDPMQALMQLLEDLVKLLK 175 P14e                  QDPAQALLQLLEDLVKLLK 176 P14f                  QDPAQALEQLLEDLVKLLK 177 P14cQAGPQSANKTGNVDDANNQDPMQALMQLLEDLVKLLK 199 P14-30                 SEEEEEALEQLLEDLVKLLK 178

It is important to note that P14d variants seem to require more sequencethan the minimal HR consensus (SEQ ID NO: 93) to HR elicitation. Inparticular, the additional C-terminal lysine residue seems to berequired for activity. This may be due to the presence of a singlehydrophilic residue between the hydrophobic repeats (LVKLL).

Example 15 Derivatives of Peptides P15/P20 that Cause HR Response inTobacco

HR tests (described in Example 1) were run on variants of P15/P20 todetermine the minimal sequence required for HR and to identify residuesof importance. The following peptides of Table 18 were determined to bepositive for HR.

TABLE 18 Peptide SEQ name Sequence ID NO: P15aNFGTPDSTVQNPQDASKPNDSQSNIAKLISALIMSLLQM  63 P15b                KPNDSQSNIAKLISALIMSLLQ  49 P20  GTPDSTVQNPQDASKPNDSQSNIAKLIS_LIMSLL  65 P15-8D-18E                KPNDSQSDIAKLISALIESLLQ  50 P15-dN4                    SQSNIAKLISALIMSLLQ 227 P15a-39PNFGTPDSTVQNPQDASKPNDSQSNIAKLISALIMSLLQP 143 P15a-34QNFGTPDSTVQNPQDASKPNDSQSNIAKLISALIQSLLQM 144 p15-59                 SEEEEEEIAKLISALIESLLE 150

Example 16 Derivatives of Peptides P17/P18 that Cause HR Response inTobacco

HR tests (described in Example 1) were run on variants of P17 and P18 todetermine the minimal sequence required for HR and to identify residuesof importance. The following peptides of Table 19 were determined to bepositive for HR.

TABLE 19 Peptide SEQ Name Sequence ID NO: P17[*]QQPIDRQTIEQMAQLLAQLLKSLL  81 P18    QQPIDRQTIEQMAQLLAQLLKSLLSPQ  83P18-1    QQPIDRQTIEQMAQLLAQLLKSLL 163 P18-2    QQPIDRQTIEQLAQLLAQLLKSLL229 P18-3    QQPIDRQTIEQLAQLLAQLLKSLLSP 228 P18-4       DRQTIEQLAQLLAQLLKSLLSP 164 P18-5          QTIEQLAQLLAQLLKSLLSP165 P18-6      SEEEEEIEQLAQLLAQLLKSLL 166 P18-7        SEEEEELAQLLAQLLKSLL 167 P18-10         SEEEEELAELLAELLKSLL 231[*] = N-terminal sequence of TSSSPGLFQSGGDNGLGGHNANSALG

Example 17 Derivatives of Peptides P19 that Cause HR Response in Tobacco

HR tests (described in Example 1) were run on variants of P19 todetermine the minimal sequence required for HR and to identify residuesof importance. The following peptides of Table 20 were determined to bepositive for HR.

TABLE 20 Peptide SEQ Name Sequence ID NO: P19 ITPDGQGGGQIGDNPLLKAMLKLIA 89 P19-20L ITPDGQGGGQIGDNPLLKALLKLIA  90 P19aITPDGQGGGQIGDNPLLKAMLKLIARMMDG  91 P19a-allLITPDGQGGGQIGDNPLLKALLKLIARLLDG  92 P19-4      QGGGQIGDNPLLKAMLKLIARMMDG226 P19-7         SEEEEEELLKALLKLIARLL 172 P19-8          SEEEEELKALLKLIARLL 173 P19-11     SEEEEEIGDNPLLKALLKLIARLL 171

It is important to note that although P19 and P19-1 exhibit HR, they donot completely conform to the consensus HR-box sequence (SEQ ID NO: 93).However, the addition of context sequence in P19-2 and P19-3 leads to asequence that does conform to the consensus. It is likely that theadditional isoleucine residues in P19, and P19-1 (N-terminal isoleucineand the IGDN sequence) increase the propensity for HR elicitation.

Example 18 Induced Resistance of Tobacco to Infection with TobaccoMosaic Virus

Peptides were tested for the induction of resistance to tobacco mosaicvirus (TMV) in tobacco. Briefly, three tobacco plants at 6-8 weeks oldwere selected per group (samples and controls). The bottom-most leaf ofthe plant was covered and the plant was sprayed with a solution of water(negative control), peptide, or Proact (positive control). The spray wasapplied until the leaves were fully wetted, indicated by liquid drippingfrom the leaves. The plants were then allowed to dry and the leafcovering was removed.

Three days post-treatment, the previously-covered leaf and a leaf on theopposite side of the plant were then lightly dusted with diatomaceousearth and 20 ul of a 1.7 ug/ml solution of purified tobacco mosaic viruswas applied. The TMV solution was then spread across the leaf surface bylightly rubbing solution and the diatomaceous earth across the surfaceof the leaves. Two minutes after inoculation, the diatomaceous earth wasrinsed off the leaves with water. 3 days after TMV inoculation, theleaves were scored based on the number of TMV lesions observed. The leafwas also scored for signs of the hypersensitive response, includingyellowing and wilting of the affected leaves.

Effectiveness described in Table 21 refers to the % decline in TMVlesions on treated vs UTC plants. A reduction of TMV on covered leavesindicates a systemic immune response in the plant while reduction onuncovered leaves indicates a local response. Asterisks indicate that theP-value derived from a T-test was <0.05.

TABLE 21 Summary of TMV Resistance SEQ ID Concentration EffectivenessEffectiveness Peptide NO: (ug/ml) Uncovered (%) Covered (%) P1 4 20 100*91* P1-allE 46 5 74* 66* polyE-min3p1 141 10 92* 79* P4 5 20 88* 89*P4-14S 6 20 80* 97* polyE-min3p4 33 10 88* 86* P6 68 20 99* 93* P6a 6720 72* 58  P14d 175 5 93* 96*

TABLE 21 Summary of TMV Resistance SEQ ID Concentration EffectivenessEffectiveness Peptide NO: (ug/ml) Uncovered (%) Covered (%) P14e 176 1090  79* P14f 177 10 72  86* P14-30 178 10 74  25  P15 64 10 95* 72* P15a63 10 88  55  P18 83 5 63* 80* P18-6 166 10 69* 86* P18-7 167 20 15* 30*P18-10 231 20 84* 87* P19 89 5 79* 90* P19-7 172 10 88* 82* P19-8 173 2074* 77* P25 182 20 94* 94* P25-11 188 10 100*  97* P30-2 189 10 94* 65*P30-3 190 10 95* 95*

In general, peptides that elicit a hypersensitive response in tobaccoalso confer a strong resistance to TMV. The peptides provided resistancein the leaves that received the treatment. However, the peptides alsocaused “system acquired resistance” whereby an immune response in onepart of a plant triggers signaling that increases immunity in otherparts of the plant. This was shown by the reduced TMV infection incovered leaves that did not directly receive the peptide treatment.Peptides that caused particularly strong immune responses included someof the minimal HR box peptide sequences: P14d, P25-11, and P30-3.

Example 19 Effect of Peptide Seed Treatment on Root and Shoot Growth

Peptides were tested for biological effects on the allocation of growthresources to the shoot (above ground) and root (below ground). Peptideswere dissolved at 0.2, 2, or 5 μg/ml in a total volume of 100 mldeionized water. Corn or soybean seeds were then soaked for one hour inthe peptide solution. Untreated control (UTC) plants were soaked indeionized water. Clear plastic 300 ml beverage cups (Solo®, DartContainer Corporation) were prepared for planting by marking the bottomwith a cross, dividing the bottom into four equal quadrants. The cupswere then filled with Sunshine Mix #1 soil (SunGro Horticulture) sievedto ¼″. 100 ml of water was added to the soil. Treated seeds were thenplanted by pressing the seed lightly into the top of the soil. The seedswere then covered with an additional 50 ml of loose soil. Seeds wereallowed to germinate and grow for 12-14 days.

The length of the shoot was measured as the distance from the soil tothe lightly stretched tip of the highest leaf for each plant. Plantsthat failed to germinate or exhibited stunted growth were removed fromthe trial. Stunting was defined as lacking a fully expanded true leaf attime of data collection or having an expanded true leaf judged by eye tobe <½ the average leaf area of the treatment group. Generally, 30 seedswere planted per treatment group and 15-25 plants were used for datacollection.

Root growth was estimated by counting the number of times that a primaryroot crosses the quadrant marks on the bottom of the cup. These wereoften observed along the bottom circumference of the cup, although somewere visible along the side of the container and were counted as ifcrossing a vertical extension of the quadrant line. This number wasdivided by 4 to produce a root growth index. This index was found tocorrelate ˜90% with measured total primary root length (sum of lengthsof all primary roots after rinsing soil from roots and measuringdirectly).

TABLE 22 Summary of Root & Shoot Growth Peptide (Host) SEQ ID NO: Rate(μg/ml) Root (%) Shoot (%) P4-14s (soy) 6 0.2 13.5* −0.1 P14c (soy) 1995.0 14.4* 9.0* P15a (corn) 63 0.2 14.2* −4.5 P15b (soy) 49 5.0 34*  15.6* P18 (corn) 83 2.0 7.7 −2.0 P30-3 (corn) 190 0.2 15.4* −0.7 P30-3(soy) 190 2.0 −17.9*  11.4* P30-3 (soy) 190 0.2 4.2 8.8* P15-59 (corn)150 0.2 7.6 −8.6* P19-8 (corn) 89 5.0 4.1 −2.7

Having thus described the basic concept of the invention, it will berather apparent to those skilled in the art that the foregoing detaileddisclosure is intended to be presented by way of example only, and isnot limiting. Various alterations, improvements, and modifications willoccur and are intended to those skilled in the art, though not expresslystated herein. These alterations, improvements, and modifications areintended to be suggested hereby, and are within the spirit and scope ofthe invention. Additionally, the recited order of processing elements orsequences, or the use of numbers, letters, or other designationstherefore, is not intended to limit the claimed processes to any orderexcept as may be specified in the claims. Accordingly, the invention islimited only by the following claims and equivalents thereto.

What is claimed:
 1. An isolated peptide comprising the amino acidsequence of(L/I/V/F)-X—X-(L/I/V/F)-(L/I)-X—X-(L/I/V/F)-(L/I/V/A)-X—X-(L/I)-(L/I/V/F)(SEQ ID NO: 93) wherein the peptide is free of cysteine and methionine;each X at positions 2, 6, and 10 is optional and, when present, is anyamino acid; and each X at positions 3, 7, and 11 is any amino acid. 2.The isolated peptide according to claim 1, wherein the peptide is lessthan 100 amino acids in length.
 3. The isolated peptide according toclaim 2, wherein the peptide is between 13 and 50 amino acids in length.4. The isolated peptide according to claim 1, wherein the isolatedpeptide is stable when dissolved in water or aqueous solution.
 5. Theisolated peptide according to claim 1, wherein the isolated peptide isresistant to chemical degradation when dissolved in an aqueous buffersolution containing a biocide.
 6. The isolated peptide according toclaim 1, wherein the isolated peptide has a solubility of greater thanabout 0.1% in water or aqueous solution.
 7. The isolated peptideaccording to claim 1, wherein one or both of X at positions 2 and 6 arenot present.
 8. The isolated peptide according to claim 1, wherein bothof X at positions 2 and 6 is present.
 9. The isolated peptide accordingto claim 1 or 7, wherein X at position 10 is not present.
 10. Theisolated peptide according to claim 1 or 8, wherein X at position 10 ispresent.
 11. The isolated peptide according to claim 1, wherein each Xat positions 2 and 6, when present, is a polar or charged amino acid;and each X at positions 3, 7, 10, and 11 is a polar or charged aminoacid.
 12. The isolated peptide according to claim 1, wherein each X atpositions 2 and 6, when present, is selected from the group consistingof R, K, D, isoD, E, N, Q, H, S, T, Y, W, G, A, and g-glutamate; andeach X at positions 3, 7, 10, and 11 is selected from the groupconsisting of R, K, D, isoD, E, N, Q, H, S, T, Y, W, G, A, andg-glutamate.
 13. The isolated peptide according to claim 1, wherein eachX at positions 2 and 6, when present, is selected from the groupconsisting of D, isoD, E, and g-glutamate; and each X at positions 3, 7,10, and 11 is selected from the group consisting of D, isoD, E, andg-glutamate.
 14. The isolated peptide according to claim 13, wherein thepeptide comprises the amino acid sequence of: SEQ ID NO: 189SEELEELLEELIEELL,, SEQ ID NO: 190 LEELLEELIEELLEE,, SEQ ID NO: 210LEELLEELIEELL,, SEQ ID NO: 213 LEELLEELLEELLEE,, SEQ ID NO: 215LEQLLEDLVELLEEE,, SEQ ID NO: 216 LEELLEDLVELLEEE,, SEQ ID NO: 217LEELLEELVELLEEE,, SEQ ID NO: 218 LEELLELFEEILEELFEE,, SEQ ID NO: 219LEELLKLFEEILEELFEE,, SEQ ID NO: 220 IEELIELIEELLEE,, SEQ ID NO: 221IEELIEELIEELLEE,, SEQ ID NO: 223 LEELLELIERLLEE,, or SEQ ID NO: 225LEELLELIEELLEE,.


15. The isolated peptide according to claim 1 further comprising ahydrophilic amino acid sequence at either the N-terminal or C-terminalend of SEQ ID NO:
 93. 16. The isolated peptide according to claim 15,wherein the peptide comprises the amino acid sequence of one of SEQ IDNOS: 31, 33, 126, 134, 141, 149, 150, 161, 166, 167, 168-173, 178,187-189, 200-202, 206-209, 231 or
 232. 17. The isolated peptideaccording to claim 1, wherein the peptide comprises the amino acidsequence of: (SEQ ID NO: 33) SEEEEELDQLLSQLIQALL;or residues 7-19 thereof (SEQ ID NO: 94) KQLDQLLSQLIQALLQP;(SEQ ID NO: 95) EKQLDQLLSQLIQALLQP; (SEQ ID NO: 96) SEKQLDQLLSQLIQALLQP;(SEQ ID NO: 97) GISEKQLDQLLSQLIQALLQP; (SEQ ID NO: 132)SQGISEKQLQLLSQLIQALLQP; (SEQ ID NO: 133) SQGISEKQLDQLLQLIQALLQP;(SEQ ID NO: 140) SQGISEKQALDQLLSQLIQALLQP; (SEQ ID NO: 141)SEEEEELDQLLTQLIEALL; or residues 7-19 thereof (SEQ ID NO: 150)SEEEEEIAKLISALIESLLE; or residues 7-20 thereof (SEQ ID NO: 167)SEEEEELAQLLAQLLKSLL; or residues 7-19 thereof (SEQ ID NO: 173)SEEEEELKALLKLIARLL; or residues 7-18 thereof (SEQ ID NO: 178)SEEEEEALEQLLEDLVKLLK; or residues 7-20 thereof (SEQ ID NO: 188)SEEEEELTGVLQKLLKILEAL; or residues 7-21 thereof (SEQ ID NO: 189)SEELEELLEELIEELL; (SEQ ID NO: 190) LEELLEELIEELLEE; (SEQ ID NO: 200)SEEEEELTLTGVLQKLLKILEA; or residues 7-22 thereof (SEQ ID NO: 201)SEEEEEVLQKLLKILEALV; or residues 7-19 thereof (SEQ ID NO: 202)SEEEEELQKLLKILEALVQ; or residues 7-19 thereof (SEQ ID NO: 210)LEELLEELIEELL; (SEQ ID NO: 213) LEELLEELLEELLEE; (SEQ ID NO: 215)LEQLLEDLVELLEEE; (SEQ ID NO: 216) LEELLEDLVELLEEE; (SEQ ID NO: 217)LEELLEELVELLEEE; (SEQ ID NO: 218) LEELLELFEEILEELFEE; (SEQ ID NO: 219)LEELLKLFEEILEELFEE; (SEQ ID NO: 220) IEELIELIEELLEE; (SEQ ID NO: 221)IEELIEELIEELLEE; (SEQ ID NO: 223) LEELLELIERLLEE; (SEQ ID NO: 225)LEELLELIEELLEE; (SEQ ID NO: 214) LEQLLEDLVKLLKEE; (SEQ ID NO: 222)LEELLKLIERLLEE; or (SEQ ID NO: 224) LEELLKLIEELLEE.


18. An isolated peptide comprising the amino acid sequence ofXXGISEKXXXXXXXXXXXXXXXX (SEQ ID NO: 1, P1/P4 consensus), wherein X atposition 1 is optional and can be S, N, D, isoD, G, A, or S; X atposition 2 is optional and can be Q, E, g-glutamate, G, A, or S; X atposition 8 is Q, E, g-glutamate, G, A, or S; X at position 9 is L, I, F,or V; X at position 10 is optional and can be D or isoD; X at position11 is Q, E, g-glutamate, G, A, or S; X at position 12 is M, L, I, or F;X at position 13 is M, L, or I; X at position 14 is optional and can beany hydrophilic amino acid; X at position 15 is Q, E, g-glutamate, G, A,S, K, or I; X at position 16 is M, L, I, V, or F; X at position 17 is M,L, I, A, or V; X at position 18 is Q, E, g-glutamate, G, A, S, M, T, orK; X at position 19 is A, D, isoD, S, V, T, K, R, E, H, or G; X atposition 20 is M, L, or I; X at position 21 is M, L, I, V, S, or F; X atposition 22 is Q, E, g-glutamate, G, A, S; X at position 23 is P, Q, E,g-glutamate, G, A, or S; and wherein the isolated peptide comprises oneor more mutations, relative to a corresponding wildtype amino acidsequence, which one or more mutations improve the stability orresistance to chemical degradation of the isolated peptide relative to apolypeptide comprising the corresponding wildtype amino acid sequence.19. The isolated peptide according to claim 18, wherein the peptide doesnot consist of NQGISEKQLDQLLTQLIMALLQQ (P1, SEQ ID NO: 4).
 20. Theisolated peptide according to claim 18, wherein the peptide does notconsist of SQGISEKQLDQLLCQLIQALLQP (P4, SEQ ID NO: 5).
 21. The isolatedpeptide according to claim 18, wherein the polypeptide comprising thecorresponding wildtype amino acid sequence consists of SEQ ID NO: 4 or5, and the isolated peptide is more stable than the polypeptide of SEQID NO: 4 or 5 when dissolved in water or aqueous solution.
 22. Theisolated peptide according to claim 18, wherein the polypeptidecomprising the corresponding wildtype amino acid sequence is SEQ ID NO:4 or 5, and the isolated peptide is more resistant to chemicaldegradation than the polypeptide of SEQ ID NO: 4 or 5 when dissolved inan aqueous buffer solution containing a biocide.
 23. The isolatedpeptide according to claim 18, wherein the peptide comprises the aminoacid sequence of SXGISEKXXDXXXXXXXXAXXXP (SEQ ID NO: 2, P4 consensus),wherein X at position 2 is Q, E, g-glutamate, G, A, or S; X at position8 is Q, E, g-glutamate, G, A, or S; X at position 9 is L, A, D, isoD, I,V, or F; X at position 11 is Q, E, g-glutamate, G, A, or S; X atposition 12 is L, D, isoD, I, or F; X at position 13 is L, I, V, or F; Xat position 14 is any hydrophilic amino acid; X at position 15 is Q, E,g-glutamate, G, A, S, K, or I; X at position 16 is L, A, I, V, M, or F;X at position 17 is I, S, or F; X at position 18 is Q, E, g-glutamate,G, A, or S; X at position 20 is L, I, V, or F; X at position 21 is L orF; and X at position 22 is Q, E, g-glutamate, G, A, or S.
 24. Theisolated peptide according to claim 23, wherein X at position 14 is S.25. The isolated peptide according to claim 23, wherein the peptidecomprises the amino acid sequence of one of SEQ ID NOS: 6-11, 19, 20,22-24, 27-30, 32, 34, 37, 40, 98-108, 111-114, 116, 117, 119-125,127-131, 136-139, 191, and
 196. 26. The isolated peptide according toclaim 18, wherein the peptide comprises the amino acid sequence ofXXGISEKXLDXLLTXLIXALLXX (SEQ ID NO: 3, P1 consensus), wherein X atposition 1 is N, D, isoD, G, A, or S; X at position 2 is Q, E,g-glutamate, G, A, or S; X at position 8 is Q, E, g-glutamate, G, A, orS; X at position 11 is Q, E, g-glutamate, G, A, or S; X at position 15is Q, E, g-glutamate, G, A, or S; X at position 18 is M, T, K, E,g-glutamate, G, A, or S; X at position 22 is Q, E, g-glutamate, G, A, orS; and X at position 23 is Q, E, g-glutamate, G, A, or S.
 27. Theisolated peptide according to claim 26, wherein X is E, g-glutamate, G,A, or S for at least one of positions 2, 8, 11, 15, 22, and 23 of SEQ IDNO:
 3. 28. The isolated peptide according to claim 26, wherein thepeptide comprises the amino acid sequence of one of SEQ ID NOS: 41-46,109, 110, 115, and
 118. 29. The isolated peptide according to claim 18,wherein the peptide is less than 100 amino acids in length.
 30. Theisolated peptide according to claim 29, wherein the peptide is between23 and 50 peptides in length.
 31. The isolated peptide according toclaim 18, wherein the peptide consists essentially of the recited aminoacid sequence.
 32. The isolated peptide according to claim 18, whereinthe peptide consists of the recited amino acid sequence.
 33. An isolatedpeptide of less than 100 amino acids in length, which isolated peptidecomprises the amino acid sequence of SEQ ID NO:
 5. 34. The isolatedpeptide according to claim 33, wherein the isolated peptide consistsessentially of SEQ ID NO:
 5. 35. The isolated peptide according to claim33, wherein the isolated peptide consists of SEQ ID NO:
 5. 36. Anisolated peptide comprising the amino acid sequence of (i)KPXDSXSXIAKLISXLIXSLLX (SEQ ID NO: 47, P15b/P20 consensus), wherein X atposition 3 is N, D, or isoD; X at position 6 is Q, E, g-glutamate, G, A,or S; X at position 8 is N, D, or isoD; X at position 15 is optional andcan be any amino acid; X at position 18 is M, E, g-glutamate, G, A, S,T, or K; and X at position 22 is optional and can be Q, E, g-glutamate,G, A, or S; or (ii) IAKLISXLIXSLLX (SEQ ID NO: 12, P15/20 minconsensus), wherein X at position 7 is optional and can be any aminoacid; X at position 10 is M, E, g-glutamate, G, A, S, T, or K; and X atposition 14 is optional and can be Q, E, g-glutamate, G, A, or S.wherein the isolated peptide comprises one or more mutations, relativeto a corresponding wildtype amino acid sequence, which one or moremutations improve the stability or resistance to chemical degradation ofthe isolated peptide relative to a polypeptide comprising thecorresponding wildtype amino acid sequence.
 37. The isolated peptideaccording to claim 36, wherein the isolated peptide is less than 100amino acids in length.
 38. The isolated peptide according to claim 36,wherein the polypeptide comprising the corresponding wildtype amino acidsequence is SEQ ID NO: 48, and the isolated peptide is more stable thanthe polypeptide of SEQ ID NO: 48 when dissolved in water or aqueoussolution.
 39. The isolated peptide according to claim 36, wherein thepolypeptide comprising the corresponding wildtype amino acid sequence isSEQ ID NO: 48, and the isolated peptide is more resistant to chemicaldegradation than the polypeptide of SEQ ID NO: 48 when dissolved in anaqueous buffer solution containing a biocide.
 40. The isolated peptideaccording to claim 36, wherein the peptide comprises the amino acidsequence of one of SEQ ID NOS: 49-65, 142-144, 151, or
 152. 41. Theisolated peptide according to claim 36, wherein the peptide consistsessentially of the recited amino acid sequence.
 42. The isolated peptideaccording to claim 36, wherein the peptide consists of the recited aminoacid sequence.
 43. An isolated peptide comprising from 22 to 36 aminoacids, wherein the peptide consists essentially of the amino acids ofSEQ ID NO: 49, SEQ ID NO: 63, or SEQ ID NO:
 64. 44. An isolated peptidecomprising the amino acid sequence of SEQ ID NO:
 65. 45. An isolatedpeptide comprising the amino acid sequence of (i) PSPXTXXLXXIVGXILXAXN(SEQ ID NO: 66, peptide 6/6a consensus), wherein X at position 4 is F orY; X at position 6 is Q, E, g-glutamate, G, A, or S; X at position 7 isoptional and can be M, E, g-glutamate, G, A, S, T, or K; X at position 9is M, E, g-glutamate, G, A, S, T, or K; X at position 10 is H or N; X atposition 14 is E, g-glutamate, D, or isoD; X at position 17 is Q, E,g-glutamate, G, A, or S; and X at position 19 is Q, E, g-glutamate, G,A, or S; or (ii) XTXXLXXIVGXIL (SEQ ID NO: 135, P6/6a min consensus),wherein X at position 1 is F or Y; X at position 3 is Q, E, g-glutamate,G, A, or S; X at position 4 is optional and, according to oneembodiment, can be M, E, g-glutamate, G, A, S, T, or K; or according toanother embodiment can be L; X at position 6 is M, E, g-glutamate, G, A,S, T, or K; X at position 7 is H or N; and X at position 11 is E,g-glutamate, D, or isoD; wherein the isolated peptide comprises one ormore mutations relative to a corresponding wildtype amino acid sequence,which one or more mutations improve the stability or resistance tochemical degradation of the isolated peptide relative to a polypeptidecomprising the corresponding wildtype amino acid sequence.
 46. Theisolated peptide according to claim 45, wherein the peptide does notconsist of PSPFTQMLMHIVGEILQAQN (P6a, SEQ ID NO: 67).
 47. The isolatedpeptide according to claim 45, wherein the polypeptide comprising thecorresponding wildtype amino acid sequence is SEQ ID NO: 67, and theisolated peptide is more stable than the polypeptide of SEQ ID NO: 67when dissolved in water or aqueous solution.
 48. The isolated peptideaccording to claim 45, wherein the polypeptide comprising thecorresponding wildtype amino acid sequence is SEQ ID NO: 67, and theisolated peptide is more resistant to chemical degradation than thepolypeptide of SEQ ID NO: 67 when dissolved in an aqueous buffersolution containing a biocide.
 49. The isolated peptide according toclaim 45, wherein the peptide comprises the amino acid sequence of oneof SEQ ID NOS: 68-80, 155-161, 197, and
 198. 50. The isolated peptideaccording to claim 45, wherein the peptide is less 100 amino acids inlength.
 51. The isolated peptide according to claim 45, wherein thepeptide is between 20 and 50 peptides in length.
 52. The isolatedpeptide according to claim 45, wherein the peptide consists essentiallyof the recited amino acid sequence.
 53. The isolated peptide accordingto claim 45, wherein the peptide consists of the recited amino acidsequence.
 54. The isolated peptide according to claim 45, wherein thepeptide does not comprises SEQ ID NO:
 154. 55. The isolated peptideaccording to claim 45, wherein the peptide comprises the amino acidsequence of PSPXTXXLXXIVGXILXAXN (SEQ ID NO: 66, peptide 6/6aconsensus).
 56. The isolated peptide according to claim 45, wherein thepeptide comprises the amino acid sequence of XTXXLXXIVGXIL (SEQ ID NO:135, P6/6a min consensus).
 57. An isolated peptide comprising the aminoacid sequence of (i) LXXL(L/M)XILXXLV (SEQ ID NO: 16, P25 consensus)wherein X at position 2 can be Q, N, E, g-glutamate, D, isoD, T, S, A,or G; X at position 3 can be K, Q, N, E, g-glutamate, D, isoD, T, S, A,or G; X at position 6 can be K, Q, N, E, g-glutamate, D, isoD, T, S, A,or G; X at position 9 can be E, g-glutamate, D, isoD, Q, N, T, S, A, orG; and X at position 10 can be A, G, S, T, E, g-glutamate, D, isoD, Q,or N; or (ii) LXXVLXXL(L/M)XILXXLV (SEQ ID NO: 17, P25 consensus)wherein X at position 2 can be T, S, A, G, D, isoD, E, g-glutamate, Q,or N; X at position 3 can be G, T, S, A, D, isoD, E, g-glutamate, Q, orN; X at position 6 can be Q, N, E, g-glutamate, D, isoD, T, S, A, or G;X at position 7 can be K, Q, N, E, g-glutamate, D, isoD, T, S, A, or G;X at position 10 can be K, Q, N, E, g-glutamate, D, isoD, T, S, A, or G;X at position 13 can be E, g-glutamate, D, isoD, Q, N, T, S, A, or G; Xat position 14 can be A, G, S, T, E, g-glutamate, D, isoD, Q, or N; andV at position 16 is optional.
 58. The isolated peptide according toclaim 57, wherein the peptide does not consist of SEQ ID NO:
 179. 59.The isolated peptide according to claim 57, wherein the isolated peptidecomprises one or more mutations, relative to a corresponding wildtypeamino acid sequence, the polypeptide comprising the correspondingwildtype amino acid sequence is SEQ ID NO: 179, and the isolated peptideis more stable than the polypeptide of SEQ ID NO: 179 when dissolved inwater or aqueous solution.
 60. The isolated peptide according to claim57, wherein the isolated peptide comprises one or more mutations,relative to a corresponding wildtype amino acid sequence, thepolypeptide comprising the corresponding wildtype amino acid sequence isSEQ ID NO: 179, and the isolated peptide is more resistant to chemicaldegradation than the polypeptide of SEQ ID NO: 179 when dissolved in anaqueous buffer solution containing a biocide.
 61. The isolated peptideaccording to claim 57, wherein the peptide comprises the amino acidsequence of LXXL(L/M)XILXXLV (SEQ ID NO: 16, P25 consensus).
 62. Theisolated peptide according to claim 57, wherein the peptide comprisesthe amino acid sequence of LXXVLXXL(L/M)XILXXLV (SEQ ID NO: 17, P25consensus).
 63. The isolated peptide according to claim 57, wherein thepeptide comprises the amino acid sequence of LXXVLXXL(L/M)XILXXL (SEQ IDNO: 17, P25 consensus).
 64. The isolated peptide according to claim 57,wherein the peptide comprises the amino acid sequence of one of SEQ IDNOS: 180-188.
 65. The isolated peptide according to claim 57, whereinthe peptide is less 100 amino acids in length.
 66. The isolated peptideaccording to claim 57, wherein the peptide is between 20 and 50 peptidesin length.
 67. An isolated peptide comprising the amino acid sequence of(i) XXXXXXXXXXX(L/M)XXLLXXLLXXLLXXX (SEQ ID NO: 18, P17/18), wherein Xat position 1 can be any amino acid, but preferably Q, S, E,g-glutamate, A, T, G, D, isoD, N, K, or R; X at position 2 can be anyamino acid, but preferably Q, S, E, g-glutamate, A, T, G, D, isoD, N, K,or R; X at position 3 can be any amino acid, but preferably P, Q, S, E,g-glutamate, A, T, G, D, isoD, N, K, or R; X at position 4 can be anyamino acid, but preferably I, Q, S, E, g-glutamate, A, T, G, D, N, isoD,K, or R; X at position 5 can be any amino acid, but preferably D, isoD,S, E, g-glutamate, A, T, G, N, Q, K, or R; X at position 6 can be anyamino acid, but preferably R, Q, S, E, g-glutamate, A, T, G, D, isoD, N,or K; X of position 7 can be any amino acid, but preferably Q, S, E,g-glutamate, A, T, G, D, isoD, N, K, or R; X at position 8 can be anyamino acid, but preferably T, Q, S, E, g-glutamate, A, G, D, isoD, N, K,or R; X at position 9 can be any amino acid, but preferably I, Q, S, E,g-glutamate, A, T, G, D, isoD, N, K, or R; X at position 10 can be anyamino acid, but preferably E, g-glutamate, Q, S, A, T, G, D, isoD, N, K,or R; X at position 11 can be any amino acid, but preferably Q, S, E,g-glutamate, A, T, G, D, isoD, N, K, or R; X at position 13 can be anyamino acid, but preferably A, S, T, G, D, isoD, E, g-glutamate, Q, N, K,or R; X at position 14 can be any amino acid, but preferably Q, A, S, T,G, D, isoD, E, g-glutamate, N, K, or R; X at position 17 can be anyamino acid, but preferably A, S, T, G, D, isoD, E, g-glutamate, Q, N, K,or R; X at position 18 can be any amino acid, but preferably Q, A, S, T,G, D, isoD, E, g-glutamate, N, K, or R; X at position 21 can be anyamino acid, but preferably K, A, S, T, G, D, isoD, E, g-glutamate, Q, N,or R; X at position 22 can be any amino acid, but preferably S, A,T, G,D, isoD, E, g-glutamate, Q, N, K, or R; X at position 25 can be anyamino acid, but preferably S, A, T, G, D, isoD, E, g-glutamate, Q, N, K,or R; X at position 26 can be any amino acid, but preferably P, S, A, T,G, D, isoD, E, g-glutamate, Q, N, K, or R; and X at position 27 can beany amino acid, but preferably Q, S, A, T, G, D, isoD, E, g-glutamate,N, K, or R; or (ii) (L/M)XXLLXXLLXXLL (SEQ ID NO: 25, P17/18 minconsensus), wherein X at position 2 can be any amino acid, butpreferably A, S, T, G, D, isoD, E, g-glutamate, Q, N, K, or R; X atposition 3 can be any amino acid, but preferably Q, A, S, T, G, D, isoD,E, g-glutamate, N, K, or R; X at position 6 can be any amino acid, butpreferably A, S, T, G, D, isoD, E, g-glutamate, Q, N, K, or R; X atposition 7 can be any amino acid, but preferably Q, A, S, T, G, D, isoD,E, g-glutamate, N, K, or R; X at position 10 can be any amino acid, butpreferably K, A, S, T, G, D, isoD, E, g-glutamate, Q, N, or R; and X atposition 11 can be any amino acid, but preferably S, A,T, G, D, isoD, E,g-glutamate, Q, N, K, or R; wherein the isolated peptide comprises oneor more mutations relative to a corresponding wildtype amino acidsequence, and the one or more mutations improve the aqueous solubility,stability, or resistance to chemical degradation of the isolated peptiderelative to a polypeptide comprising the corresponding wildtype aminoacid sequence.
 68. The isolated peptide according to claim 67, whereinthe peptide does not consist of SEQ ID NO:
 162. 69. The isolated peptideaccording to claim 67, wherein the polypeptide comprising thecorresponding wildtype amino acid sequence is SEQ ID NO: 162, and theisolated peptide is more stable than the polypeptide of SEQ ID NO: 162when dissolved in water or aqueous solution.
 70. The isolated peptideaccording to claim 67, wherein the polypeptide comprising thecorresponding wildtype amino acid sequence is SEQ ID NO: 162, and theisolated peptide is more resistant to chemical degradation than thepolypeptide of SEQ ID NO: 162 when dissolved in an aqueous buffersolution containing a biocide.
 71. The isolated peptide according toclaim 67, wherein the peptide comprises the amino acid sequence of oneof SEQ ID NOS: 84-88,163-167, 228, 229, and
 231. 72. The isolatedpeptide according to claim 67, wherein the peptide comprises the aminoacid sequence of XXXXX XXXXX(L/M)XXLLXXLLXXLLXXX (SEQ ID NO: 18, P17/18consensus).
 73. The isolated peptide according to claim 67, wherein thepeptide comprises the amino acid sequence of (L/M)XXLLXXLLXXLL (SEQ IDNO: 25, P17/18 min consensus).
 74. The isolated peptide according toclaim 67, wherein the peptide is less 100 amino acids in length.
 75. Theisolated peptide according to claim 67, wherein the peptide is between20 and 50 peptides in length.
 76. An isolated peptide comprising theamino acid sequence of XLXX(L/M)LXLIXX(L/I/V/F/M)(L/I/V/F/M) (SEQ ID NO:26, P19 consensus), wherein X at position 1 is optional and can be L, I,V, F, or M; X at position 3 can be any amino acid, but preferably K, A,S, T, G, D, isoD, E, g-glutamate, Q, N, or R; X at position 4 can be anyamino acid, but preferably A, S, T, G, D, isoD, E, g-glutamate, Q, N, K,or R; X at position 7 can be any amino acid, but preferably K, A, S, T,G, D, isoD, E, g-glutamate, Q, N, or R; X at position 10 can be anyamino acid, but preferably A, S, T, G, D, isoD, E, g-glutamate, Q, N, K,or R; and X at position 11 can be any amino acid, but preferably R, A,S, T, G, D, isoD, E, g-glutamate, Q, N, or K.
 77. The isolated peptideaccording to claim 76, wherein the isolated peptide comprises one ormore mutations, relative to a corresponding wildtype amino acidsequence, the polypeptide comprising a corresponding wildtype amino acidsequence is SEQ ID NO: 89, and the isolated peptide is more stable thanthe polypeptide of SEQ ID NO: 89 when dissolved in water or aqueoussolution.
 78. The isolated peptide according to claim 76, wherein theisolated peptide comprises one or more mutations, relative to acorresponding wildtype amino acid sequence, the polypeptide comprisingthe corresponding wildtype amino acid sequence is SEQ ID NO: 89, and theisolated peptide is more resistant to chemical degradation than thepolypeptide of SEQ ID NO: 89 when dissolved in an aqueous buffersolution containing a biocide.
 79. The isolated peptide according toclaim 76, wherein the peptide comprises the amino acid sequence of oneof SEQ ID NOS: 90-92, 168-173, and
 226. 80. The isolated peptideaccording to claim 76, wherein the peptide is less 100 amino acids inlength.
 81. The isolated peptide according to claim 76, wherein thepeptide is between 20 and 50 peptides in length.
 82. The isolatedpeptide according to claim 76, wherein the amino acid residue atposition 1 is present.
 83. An isolated peptide comprising the amino acidsequence of (i) LXXLLXXLVXLLK (SEQ ID NO: 13, P14d consensus), wherein Xat position 1 can be: Q, N, D, E, g-glutamate, isoD, or S; X at position2 can be: D, E, g-glutamate, isoD; X at position 3 can be: P, D, E,isoD, or g-glutamate; X at position 4 can be M, A, S, D, E, isoD, org-glutamate X at position 5 can be Q, E, or g-glutamate; X at position 6can be A, E, or g-glutamate; X at position 8 can be M, L, E, Q, D, N, G,A, S, isoD, or g-glutamate; X at position 9 can be Q, N, E, D, G, A, S,isoD, or g-glutamate; X at position 12 can be Q, N, E, D, G, A, S, isoD,or g-glutamate; X at position 13 can be Q, N, E, D, G, A, S, isoD, org-glutamate; and X at position 16 can be K, Q, N, E, D, R, G, A, or S;or (ii) LXXLLXXLVXLLK (SEQ ID NO: 14, P14d min consensus), wherein X atposition 2 can be M, L, E, Q, D, N, G, A, S, isoD, or g-glutamate; X atposition 3 can be Q, N, E, D, G, A, S, isoD, or g-glutamate; X atposition 6 can be Q, N, E, D, G, A, S, isoD, or g-glutamate; X atposition 7 can be Q, N, E, D, G, A, S, isoD, or g-glutamate; and X atposition 10 can be K, Q, N, E, D, R, G, A, or S.
 84. The isolatedpeptide according to claim 83, wherein the peptide does not consist ofSEQ ID NO:
 174. 85. The isolated peptide according to claim 83, whereinthe isolated peptide comprises one or more mutations, relative to acorresponding wildtype amino acid sequence, the polypeptide comprisingthe corresponding wildtype amino acid sequence is SEQ ID NO: 174, andthe isolated peptide is more stable than the polypeptide of SEQ ID NO:174 when dissolved in water or aqueous solution.
 86. The isolatedpeptide according to claim 83, wherein the isolated peptide comprisesone or more mutations, relative to a corresponding wildtype amino acidsequence, the polypeptide comprising the corresponding wildtype aminoacid sequence is SEQ ID NO: 174, and the isolated peptide is moreresistant to chemical degradation than the polypeptide of SEQ ID NO: 174when dissolved in an aqueous buffer solution containing a biocide. 87.The isolated peptide according to claim 83, wherein the peptidecomprises the amino acid sequence of one of SEQ ID NOS: 175-178 and 199.88. The isolated peptide according to claim 83, wherein the peptidecomprises the amino acid sequence of XXXXXXLXXLLXXLVXLLK (SEQ ID NO: 13,P14d consensus).
 89. The isolated peptide according to claim 83, whereinthe peptide comprises the amino acid sequence of LXXLLXXLVXLLK (SEQ IDNO: 14, Pl4d min consensus).
 90. The isolated peptide according to claim83, wherein the peptide is less 100 amino acids in length.
 91. Theisolated peptide according to claim 83, wherein the peptide is between12 and 50 peptides in length.
 92. An isolated peptide comprising theamino acid sequence of (i) (L/M)XXLLX(L/M)FXXI(L/M)XX (SEQ ID NO: 15,P3min consensus) wherein X at position 2 can be Q, N, E, g-glutamate, D,isoD, T, S, A, or G; X at position 3 can be Q, N, E, g-glutamate, D,isoD, T, S, A, or G; X at position 6 can be K, Q, N, E, g-glutamate, D,isoD, T, S, A, or G; X at position 9 can be E, g-glutamate, D, isoD, Q,N, T, S, A, or G; X at position 10 can be A, G, S, T, E, g-glutamate, D,isoD, Q, or N; X at position 13 can be Q, N, E, g-glutamate, D, isoD, T,S, A, or G; and X at position 14 can be Q, N, E, g-glutamate, D, isoD,T, S, A, or G.
 93. The isolated peptide according to claim 92, whereinthe peptide does not consist of SEQ ID NO: 230 or the C-terminal 31amino acid portion thereof.
 94. The isolated peptide according to claim92, wherein the isolated peptide comprises one or more mutations,relative to a corresponding wildtype amino acid sequence, thepolypeptide comprising the corresponding wildtype amino acid sequence isSEQ ID NO: 230, and the isolated peptide is more stable than thepolypeptide of SEQ ID NO: 230 when dissolved in water or aqueoussolution.
 95. The isolated peptide according to claim 92, wherein theisolated peptide comprises one or more mutations, relative to acorresponding wildtype amino acid sequence, the polypeptide comprisingthe corresponding wildtype amino acid sequence is SEQ ID NO: 230, andthe isolated peptide is more resistant to chemical degradation than thepolypeptide of SEQ ID NO: 230 when dissolved in an aqueous buffersolution containing a biocide.
 96. The isolated peptide according toclaim 92, wherein the isolated peptide comprises one or more mutations,relative to a corresponding wildtype amino acid sequence, thepolypeptide comprising the corresponding wildtype amino acid sequence isSEQ ID NO: 230, and the isolated peptide is more soluble in aqueoussolution than the polypeptide of SEQ ID NO:
 230. 97. The isolatedpeptide according to claim 92, wherein the peptide consists of SEQ IDNO:
 204. 98. The isolated peptide according to claim 92, wherein thepeptide comprises the amino acid sequence of one of SEQ ID NOS: 205-209.99. The isolated peptide according to claim 92, wherein the peptide isless 100 amino acids in length.
 100. The isolated peptide according toclaim 92, wherein the peptide is between 12 and 30 peptides in length.101. The isolated peptide according to one of claims 1 to 100, whereinthe peptide is at least 90% pure.
 102. The isolated peptide according toone of claims 1 to 30, 33, 36 to 40, 44 to 51, 54 to 96, and 98 to 100wherein the peptide is a fusion polypeptide comprising a second aminoacid sequence coupled via peptide bond to the amino acid sequence. 103.The isolated peptide according to claim 102, wherein the second aminoacid sequence includes a purification tag.
 104. The isolated peptideaccording to claim 103, wherein the second amino acid sequence furtherincludes a cleavable linker sequence between the purification tag andthe amino acid sequence.
 105. The isolated peptide according to claim102, wherein the peptide is a fusion polypeptide comprising a firstamino acid sequence for said peptide linked to a second amino acidsequence for said peptide.
 106. The isolated peptide according to claim102, wherein the second amino acid sequence includes an N-terminal orC-terminal hydrophilic amino acid sequence.
 107. The isolated peptideaccording to claim 106, wherein the hydrophilic amino acid sequencecomprises a plurality of Glu (E) amino acid residues.
 108. A fusionpolypeptide comprising a plurality of amino acid sequences linkedtogether in series, each of the plurality of amino acid sequencescomprising the peptide according to one of claims 1 to 30, 33, 36 to 40,44 to 51, 54 to 96, and 98 to
 100. 109. The fusion polypeptide accordingto claim 108, wherein the plurality of amino acid sequences are linkedtogether by a cleavable linker sequence.
 110. The fusion polypeptideaccording to claim 108, wherein the plurality of amino acid sequenceseach comprise a purification tag, an N-terminal or C-terminalhydrophilic amino acid sequence, or both.
 111. A composition comprisingone or more peptides according to one of claims 1 to 107 or a fusionpolypeptide according to one of claims 108 to 110, and a carrier. 112.The composition according to claim 111, wherein the composition is aclarified cell extract.
 113. The composition according to claim 111further comprising an additive selected from the group consisting offertilizer, herbicide, insecticide, fungicide, nematicide, abactericidal agent, a biological inoculant, a plant regulator, andmixtures thereof.
 114. The composition according to claim 113, whereinthe insecticide is a neonicotinoid insecticide, an organophosphateinsecticide, a pyrethroid insecticide, a macrocyclic lactoneinsecticide, a carbamate insecticide, a diamide insecticide, anavermectin insecticide, a chitin synthesis inhibitor, or any combinationthereof.
 115. The composition according to claim 113, wherein thefungicide is a strobilurin fungicide, a triazole fungicide, a succinatedehydrogenase fungicide, a phenylamide fungicide, a phenylpyrrolefungicide, a phthalimide fungicide, a dithiocarbamate fungicide, abenzimidazole fungicide, or any combination thereof.
 116. Thecomposition according to claim 113, wherein the nematicide is acarbamate nematicide.
 117. The composition according to claim 113,wherein the bactericidal agent is a dichlorophene and benzylalcohol hemiformal bactericide, an isothiazolinone bactericide, or a combinationthereof.
 118. The composition according to claim 113, wherein biologicalinoculant is a Bradyrhizobium spp., a Bacillus spp., a Streptomycesspp., a Trichoderma spp., a Pasteuria spp., or any combination thereof.119. The composition according to claim 113, wherein the compositioncomprises: one or more of peptides P1, P4-14S, P6a, P14d, P15a, P18,P19, or P25; and clothianidin, a combination of clothianidin andBacillus firmus, imidicloprid, or a combination of imidicloprid andBacillus firmus.
 120. The composition according to claim 113, whereinthe composition comprises: one or more of peptides P1, P4-14S, P6a,P14d, P15a, P18, P19, or P25; and thiamethoxam; a combination ofthiamethoxam, mefenoxam, and fludioxynil; a combination of thiamethoxam,mefenoxam, fludioxynil and azoxystrobin; a combination of thiamethoxamand abamectin; a combination of thiamethoxam, abamectin, and a Pasteurianematicide; or a combination of thiamethoxam, mefenoxam, fludioxynil,azoxystrobin, thiabendazole, and abamectin.
 121. The compositionaccording to claim 113, wherein the composition comprises: one or moreof peptides P1, P4-14S, P6a, P14d, P15a, P18, P19, or P25; and abiological inoculant comprising a Bradyrhizobium spp., a Bacillus spp.,and a combination thereof.
 122. The composition according to claim 111,wherein the carrier is an aqueous carrier.
 123. The compositionaccording to claim 122, wherein the aqueous carrier further comprisesone or more of a biocidal agent, a protease inhibitor, a non-ionicsurfactant, or a combination thereof.
 124. The composition according toclaim 111, wherein the carrier is a solid carrier in particulate form.125. The composition according to claim 124, wherein the solid carrieris a dry powder.
 126. A method of imparting disease resistance to plantscomprising: applying an effective amount of an isolated peptideaccording to one of claims 1 to 107, a fusion polypeptide according toone of claims 108 to 110, or a composition according to one of claims111 to 125 to a plant or plant seed or the locus where the plant isgrowing or is expected to grow, wherein said applying is effective toimpart disease resistance.
 127. The method according to claim 126,wherein the disease is a viral disease, a bacterial disease, or a fungaldisease.
 128. The method according to claim 126, wherein said applyingis carried out with a plant.
 129. The method according to claim 128,wherein the plant is tolerant to at least one herbicide.
 130. The methodaccording to claim 126, wherein said applying is carried out with aplant seed, said method further comprising planting the seed treatedwith the peptide or composition in natural or artificial soil, andpropagating a plant from the seed planted in the soil.
 131. The methodaccording to claim 126, wherein said applying is carried out at thelocus where the plant is growing or is expected to grow.
 132. The methodaccording to claim 126, wherein the plant is selected from agricultural,silvicultural, ornamental and horticultural plants each in its naturalor genetically modified form.
 133. The method according to claim 126,wherein the plant is a genetically modified plant.
 134. The methodaccording to claim 126, wherein the plant to be treated is selected fromthe group consisting of alfalfa, apple, apricot, asparagus, avocados,bananas, barley, beans, beech (Fagus spec.), begonia, birch, blackberry,blueberry, cabbage, camphor, canola, carrot, castor oil plant, cherry,cinnamon, citrus, cocoa bean, coffee, corn, cotton, cucumber, cucurbit,eucalyptus, fir, flax, fodder beet, fuchsia, garlic, geranium, grapes,ground nut, hemp, hop, juneberry, juncea (Brassica juncea), jute,lentil, lettuce, linseed, melon, mustard, oak, oats, oil palm, oil-seedrape, olive, onion, paprika, pea, peach, pear, pelargonium, peppers,petunia, pine (Pinus spec.), poplar (Populus spec.), pome fruit, potato,rape, raspberry, rice, rubber tree, rye, sorghum, soybean, spinach,spruce, squash, strawberry, sugar beet, sugar cane, sunflower, tea,teak, tobacco, tomato, triticale, turf, watermelon, wheat and willow(Salix spec.).
 135. A method of enhancing plant growth comprising:applying an effective amount of an isolated peptide according to one ofclaims 1 to 107, a fusion polypeptide according to one of claims 108 to110, or a composition according to one of claims 111 to 125 to a plantor plant seed or the locus where the plant is growing or is expected togrow, wherein said applying is effective to enhance plant growth. 136.The method according to claim 135, wherein the enhanced growth comprisesimproved plant vigor, increased plant weight, increased biomass,increased number of flowers per plant, higher grain and/or fruit yield,more tillers or side shoots, larger leaves, increased shoot growth,increased protein content, increased oil content, increased starchcontent, increased pigment content, increased chlorophyll content, andcombinations thereof.
 137. The method according to claim 135, whereinsaid applying is carried out with a plant.
 138. The method according toclaim 137, wherein the plant is tolerant to at least one herbicide. 139.The method according to claim 135, wherein said applying is carried outwith a plant seed, said method further comprising planting the seedtreated with the peptide or composition in natural or artificial soil,and propagating a plant from the seed planted in the soil.
 140. Themethod according to claim 135, wherein said applying is carried out atthe locus where the plant is growing or is expected to grow.
 141. Themethod according to claim 135, wherein the plant is selected fromagricultural, silvicultural, ornamental and horticultural plants each inits natural or genetically modified form.
 142. The method according toclaim 135, wherein the plant is a genetically modified plant.
 143. Themethod according to claim 135, wherein the plant to be treated isselected from the group consisting of alfalfa, apple, apricot,asparagus, avocados, bananas, barley, beans, beech (Fagus spec.),begonia, birch, blackberry, blueberry, cabbage, camphor, canola, carrot,castor oil plant, cherry, cinnamon, citrus, cocoa bean, coffee, corn,cotton, cucumber, cucurbit, eucalyptus, fir, flax, fodder beet, fuchsia,garlic, geranium, grapes, ground nut, hemp, hop, juneberry, juncea(Brassica juncea), jute, lentil, lettuce, linseed, melon, mustard, oak,oats, oil palm, oil-seed rape, olive, onion, paprika, pea, peach, pear,pelargonium, peppers, petunia, pine (Pinus spec.), poplar (Populusspec.), pome fruit, potato, rape, raspberry, rice, rubber tree, rye,sorghum, soybean, spinach, spruce, squash, strawberry, sugar beet, sugarcane, sunflower, tea, teak, tobacco, tomato, triticale, turf,watermelon, wheat and willow (Salix spec.).
 144. A method of increasinga plant's tolerance to biotic stress comprising: applying an effectiveamount of an isolated peptide according to one of claims 1 to 107, afusion polypeptide according to one of claims 108 to 110, or acomposition according to one of claims 111 to 125 to a plant or plantseed or the locus where the plant is growing or is expected to grow,wherein said applying is effective to increase the plant's tolerance tobiotic stress factors selected from the group consisting of insects,arachnids, nematodes, weeds, and combinations thereof.
 145. The methodaccording to claim 144, wherein said applying is carried out with aplant.
 146. The method according to claim 145, wherein the plant istolerant to at least one herbicide.
 147. The method according to claim144, wherein said applying is carried out with a plant seed, said methodfurther comprising planting the seed treated with the peptide orcomposition in natural or artificial soil, and propagating a plant fromthe seed planted in the soil.
 148. The method according to claim 144,wherein said applying is carried out at the locus where the plant isgrowing or is expected to grow.
 149. The method according to claim 144,wherein the plant is selected from agricultural, silvicultural,ornamental and horticultural plants each in its natural or geneticallymodified form.
 150. The method according to claim 144, wherein the plantis a genetically modified plant.
 151. The method according to claim 144,wherein the plant to be treated is selected from the group consisting ofalfalfa, apple, apricot, asparagus, avocados, bananas, barley, beans,beech (Fagus spec.), begonia, birch, blackberry, blueberry, cabbage,camphor, canola, carrot, castor oil plant, cherry, cinnamon, citrus,cocoa bean, coffee, corn, cotton, cucumber, cucurbit, eucalyptus, fir,flax, fodder beet, fuchsia, garlic, geranium, grapes, ground nut, hemp,hop, juneberry, juncea (Brassica juncea), jute, lentil, lettuce,linseed, melon, mustard, oak, oats, oil palm, oil-seed rape, olive,onion, paprika, pea, peach, pear, pelargonium, peppers, petunia, pine(Pinus spec.), poplar (Populus spec.), pome fruit, potato, rape,raspberry, rice, rubber tree, rye, sorghum, soybean, spinach, spruce,squash, strawberry, sugar beet, sugar cane, sunflower, tea, teak,tobacco, tomato, triticale, turf, watermelon, wheat and willow (Salixspec.).
 152. A method of increasing a plant's tolerance to abioticstress comprising: applying an effective amount of an isolated peptideaccording to one of claims 1 to 107, a fusion polypeptide according toone of claims 108 to 110, or a composition according to one of claims111 to 125 to a plant or plant seed or the locus where the plant isgrowing or is expected to grow, wherein said applying is effective toincrease the plant's tolerance to abiotic stress factors selected fromthe group consisting of salt stress, water stress, ozone stress, heavymetal stress, cold stress, heat stress, nutritional stress, andcombinations thereof.
 153. The method according to claim 152, whereinsaid applying is carried out with a plant.
 154. The method according toclaim 153, wherein the plant is tolerant to at least one herbicide. 155.The method according to claim 152, wherein said applying is carried outwith a plant seed, said method further comprising planting the seedtreated with the peptide or composition in natural or artificial soil,and propagating a plant from the seed planted in the soil.
 156. Themethod according to claim 152, wherein said applying is carried out atthe locus where the plant is growing or is expected to grow.
 157. Themethod according to claim 152, wherein the plant is selected fromagricultural, silvicultural, ornamental and horticultural plants each inits natural or genetically modified form.
 158. The method according toclaim 152, wherein the plant is a genetically modified plant.
 159. Themethod according to claim 152, wherein the plant to be treated isselected from the group consisting of alfalfa, apple, apricot,asparagus, avocados, bananas, barley, beans, beech (Fagus spec.),begonia, birch, blackberry, blueberry, cabbage, camphor, canola, carrot,castor oil plant, cherry, cinnamon, citrus, cocoa bean, coffee, corn,cotton, cucumber, cucurbit, eucalyptus, fir, flax, fodder beet, fuchsia,garlic, geranium, grapes, ground nut, hemp, hop, juneberry, juncea(Brassica juncea), jute, lentil, lettuce, linseed, melon, mustard, oak,oats, oil palm, oil-seed rape, olive, onion, paprika, pea, peach, pear,pelargonium, peppers, petunia, pine (Pinus spec.), poplar (Populusspec.), pome fruit, potato, rape, raspberry, rice, rubber tree, rye,sorghum, soybean, spinach, spruce, squash, strawberry, sugar beet, sugarcane, sunflower, tea, teak, tobacco, tomato, triticale, turf,watermelon, wheat and willow (Salix spec.).
 160. A method impartingdesiccation resistance to cuttings removed from ornamental plantscomprising: applying an effective amount of an isolated peptideaccording to one of claims 1 to 107, a fusion polypeptide according toone of claims 108 to 110, or a composition according to one of claims111 to 125 to a plant or the locus where the plant is growing, whereinsaid applying is effective to impart desiccation resistance to cuttingsremoved from the ornamental plant.
 161. The method according to claim160, wherein said applying is carried out with an ornamental plant. 162.The method according to claim 160, wherein said applying is carried outat the locus where the ornamental plant is growing.
 163. The methodaccording to claim 160, wherein the ornamental plant is a geneticallymodified ornamental plant.
 164. The method according to claim 160,wherein the plant to be treated is selected from the group consisting ofbeech (Fagus spec.), begonia, birch, ornamental cabbage, fir, fuchsia,garlic, geranium, oak, ornamental onion, pelargonium, petunia, pine(Pinus spec.), poplar (Populus spec.), sunflower, teak, tobacco, turf,and willow (Salix spec.).
 165. A method of imparting post-harvestdisease or post-harvest desiccation resistance to a fruit or vegetablecomprising: applying an effective amount of an isolated peptideaccording to one of claims 1 to 107, a fusion polypeptide according toone of claims 108 to 110, or a composition according to one of claims111 to 125 to a plant containing a fruit or vegetable or the locus wherethe plant is growing, or applying an effective amount of the isolatedpeptide or the composition to a harvested fruit or vegetable, whereinsaid applying is effective to impart post-harvest disease resistance ordesiccation resistance to the fruit or vegetable.
 166. The methodaccording to claim 165, wherein said applying is carried out with aplant.
 167. The method according to claim 166, wherein the plant istolerant to at least one herbicide.
 168. The method according to claim165, wherein said applying is carried out at the locus where the plantis growing.
 169. The method according to claim 165, wherein saidapplying is carried out with a harvested fruit or vegetable.
 170. Themethod according to claim 165, wherein the plant is a geneticallymodified plant.
 171. The method according to claim 165, wherein theplant is selected from the group consisting of apple, apricot,asparagus, avocados, bananas, blackberry, blueberry, cabbage, carrot,cherry, citrus, corn, cucumber, cucurbit, fodder beet, garlic, grapes,juneberry, juncea (Brassica juncea), lettuce, melon, mustard, olive,onion, pea, peach, pear, peppers, pome fruit, potato, rape, raspberry,spinach, squash, strawberry, sugar beet, sugar cane, tea, tomato,triticale, and watermelon.
 172. A method of enhancing the longevity offruit or vegetable ripeness comprising: applying an effective amount ofan isolated peptide according to one of claims 1 to 107, a fusionpolypeptide according to one of claims 108 to 110, or a compositionaccording to one of claims 111 to 125 to a plant containing a fruit orvegetable or the locus where the plant is growing, or applying aneffective amount of the isolated peptide or the composition to aharvested fruit or vegetable, wherein said applying is effective toenhance the longevity of fruit or vegetable ripeness.
 173. The methodaccording to claim 172, wherein said applying is carried out with aplant.
 174. The method according to claim 172, wherein the plant istolerant to at least one herbicide.
 175. The method according to claim172, wherein said applying is carried out at the locus where the plantis growing.
 176. The method according to claim 172, wherein saidapplying is carried out with a harvested fruit or vegetable.
 177. Themethod according to claim 172, wherein the plant is a geneticallymodified plant.
 178. The method according to claim 172, wherein theplant is selected from the group consisting of apple, apricot,asparagus, avocados, bananas, blackberry, blueberry, cabbage, carrot,cherry, citrus, corn, cucumber, cucurbit, fodder beet, garlic, grapes,juneberry, juncea (Brassica juncea), lettuce, melon, mustard, olive,onion, pea, peach, pear, peppers, pome fruit, potato, rape, raspberry,spinach, squash, strawberry, sugar beet, sugar cane, tea, tomato,triticale, and watermelon.
 179. A method of modulating plant biochemicalsignaling comprising: applying an effective amount of an isolatedpeptide according to one of claims 1 to 107, a fusion polypeptideaccording to one of claims 108 to 110, or a composition according to oneof claims 111 to 125 to a plant or plant seed or the locus where theplant is growing or is expected to grow, wherein said applying iseffective to modulate plant biochemical signaling.
 180. The methodaccording to claim 179, where the plant biochemical signaling isselected from the group consisting of induction of nitric oxideproduction, peroxide production, or a secondary metabolite; agonisticmodulation of the ethylene signaling pathway and induction ofethylene-responsive gene expression; agonistic modulation of thesalicylic acid signaling pathway and induction of salicylicacid-responsive gene expression; agonistic modulation of the abscisicacid pathway and induction of abscisic acid-responsive gene expression;agonistic modulation of the gibberellin signaling pathway and inductionof gibberellin-responsive gene expression; antagonistic modulation ofjasmonic acid signaling and inhibiting expression of jasmonicacid-responsive genes; inducing protease inhibitor expression; inducingreactive oxygen species production in plant tissues; inducingimmune-related and antimicrobial peptide production; and inducingexpansin gene expression and production.
 181. A DNA construct comprisinga first nucleic acid molecule encoding a polypeptide isolated peptideaccording to one of claims 1 to 107 or a fusion polypeptide according toone of claims 108 to 110, and a promoter-effective nucleic acid moleculeoperably coupled to the first nucleic acid molecule.
 182. A recombinantexpression vector comprising the DNA construct according to claim 181.183. A recombinant host cell comprising the DNA construct according toclaim
 181. 184. The recombinant host cell according to claim 183,wherein the recombinant host cell is a plant protoplast.
 185. Therecombinant host cell according to claim 183, wherein the recombinanthost cell is a bacterium.
 186. A transgenic plant comprising arecombinant host cell according to claim
 183. 187. A transgenic plantseed comprising a recombinant host cell according to claim
 183. 188. Atransgenic plant comprising the DNA construct according to claim 181.189. A transgenic plant seed comprising the DNA construct according toclaim
 181. 190. A method of imparting disease resistance to plantscomprising: providing a transgenic plant transformed with the DNAconstruct according to claim 181; and growing the plant under conditionseffective to permit the DNA construct to express the peptide or thefusion polypeptide to impart disease resistance.
 191. A method ofenhancing plant growth comprising: providing a transgenic planttransformed with the DNA construct according to claim 181; and growingthe plant under conditions effective to permit the DNA construct toexpress the peptide or the fusion polypeptide to enhance plant growth.192. A method of imparting disease resistance to plants comprising:providing a transgenic plant transformed with the DNA constructaccording to claim 181; and growing the plant under conditions effectiveto permit the DNA construct to express the peptide or the fusionpolypeptide to impart disease resistance.
 193. A method of increasing aplant's tolerance to biotic stress comprising: providing a transgenicplant transformed with the DNA construct according to claim 181; andgrowing the plant under conditions effective to permit the DNA constructto express the peptide or the fusion polypeptide to increase the plant'stolerance to biotic stress factors selected from the group consisting ofinsects, arachnids, nematodes, weeds, and combinations thereof.
 194. Amethod of increasing a plant's tolerance to abiotic stress comprising:providing a transgenic plant transformed with the DNA constructaccording to claim 181; and growing the plant under conditions effectiveto permit the DNA construct to express the peptide or the fusionpolypeptide to increase the plant's tolerance to abiotic stress factorsselected from the group consisting of salt stress, drought stress, ozonestress, heavy metal stress and cold stress, and combinations thereof.195. A method of imparting desiccation resistance to cuttings removedfrom ornamental plants comprising: providing a transgenic ornamentalplant transformed with the DNA construct according to claim 181; andgrowing the plant under conditions effective to permit the DNA constructto express the peptide or the fusion polypeptide to impart desiccationresistance to cuttings removed from the transgenic ornamental plant.196. A method of imparting post-harvest disease or post-harvestdesiccation resistance to a fruit or vegetable comprising: providing atransgenic plant transformed with the DNA construct according to claim181; and growing the plant under conditions effective to permit the DNAconstruct to express the peptide or the fusion polypeptide to impartpost-harvest disease resistance or desiccation resistance to a fruit orvegetable removed from the transgenic plant.
 197. A method of enhancingthe longevity of fruit or vegetable ripeness comprising: providing atransgenic plant transformed with the DNA construct according to claim181; and growing the plant under conditions effective to permit the DNAconstruct to express the peptide or the fusion polypeptide to enhancelongevity of ripeness for a fruit or vegetable removed from thetransgenic plant.
 198. A method of imparting disease resistance toplants comprising: providing a transgenic plant seed transformed withthe DNA construct according to claim 181; planting the transgenic plantseed in soil; and propagating a transgenic plant from the transgenicplant seed to permit the DNA construct to express the peptide or thefusion polypeptide to impart disease resistance.
 199. A method ofenhancing plant growth comprising: providing a transgenic plant seedtransformed with the DNA construct according to claim 181; planting thetransgenic plant seed in soil; and propagating a transgenic plant fromthe transgenic plant seed to permit the DNA construct to express thepeptide or the fusion polypeptide to enhance plant growth.
 200. A methodof imparting disease resistance to plants comprising: providing atransgenic plant seed transformed with the DNA construct according toclaim 181; planting the transgenic plant seed in soil; and propagating atransgenic plant from the transgenic plant seed to permit the DNAconstruct to express the peptide or the fusion polypeptide to impartdisease resistance.
 201. A method of increasing a plant's tolerance tobiotic stress comprising: providing a transgenic plant seed transformedwith the DNA construct according to claim 181; planting the transgenicplant seed in soil; and propagating a transgenic plant from thetransgenic plant seed to permit the DNA construct to express the peptideor the fusion polypeptide to increase the plant's tolerance to bioticstress factors selected from the group consisting of insects, arachnids,nematodes, weeds, and combinations thereof.
 202. A method of increasinga plant's tolerance to abiotic stress comprising: providing a transgenicplant seed transformed with the DNA construct according to claim 181;planting the transgenic plant seed in soil; and propagating a transgenicplant from the transgenic plant seed to permit the DNA construct toexpress the peptide or the fusion polypeptide to increase the plant'stolerance to abiotic stress factors selected from the group consistingof salt stress, drought stress, ozone stress, heavy metal stress andcold stress, and combinations thereof.
 203. A method of impartingdesiccation resistance to cuttings removed from ornamental plantscomprising: providing a transgenic ornamental plant seed transformedwith the DNA construct according to claim 181; planting the transgenicornamental plant seed in soil; and propagating a transgenic ornamentalplant from the transgenic ornamental plant seed to permit the DNAconstruct to express the peptide or the fusion polypeptide to impartdesiccation resistance to cuttings removed from the transgenicornamental plant.
 204. A method of imparting post-harvest disease orpost-harvest desiccation resistance to a fruit or vegetable comprising:providing a transgenic plant seed transformed with the DNA constructaccording to claim 181; planting the transgenic plant seed in soil; andpropagating a transgenic plant from the transgenic plant seed to permitthe DNA construct to express the peptide or the fusion polypeptide toimpart post-harvest disease resistance or desiccation resistance to afruit or vegetable removed from the transgenic plant.
 205. A method ofenhancing the longevity of fruit or vegetable ripeness comprising:providing a transgenic plant seed transformed with the DNA constructaccording to claim 181; planting the transgenic plant seed in soil; andpropagating a transgenic plant from the transgenic plant seed to permitthe DNA construct to express the peptide or the fusion polypeptide toenhance longevity of ripeness for a fruit or vegetable removed from thetransgenic plant.
 206. A method of modulating plant biochemicalsignaling comprising: providing a transgenic plant seed transformed withthe DNA construct according to claim 181; planting the transgenic plantseed in soil; and propagating a transgenic plant from the transgenicplant seed to permit the DNA construct to express the peptide or thefusion polypeptide to modulate plant biochemical signaling.
 207. Themethod according to claim 206, where the plant biochemical signaling isselected from the group consisting of induction of nitric oxideproduction, peroxide production, or a secondary metabolite; agonisticmodulation of the ethylene signaling pathway and induction ofethylene-responsive gene expression; agonistic modulation of thesalicylic acid signaling pathway and induction of salicylicacid-responsive gene expression; agonistic modulation of the abscisicacid pathway and induction of abscisic acid-responsive gene expression;agonistic modulation of the gibberellin signaling pathway and inductionof gibberellin-responsive gene expression; antagonistic modulation ofjasmonic acid signaling and inhibiting expression of jasmonicacid-responsive genes; inducing protease inhibitor expression; inducingreactive oxygen species production in plant tissues; inducingimmune-related and antimicrobial peptide production; and inducingexpansin gene expression and production.